Alkaloids: an Overview of Their Antibacterial, Antibiotic-Enhancing and Antivirulence Activities

International Journal of Antimicrobial Agents 44 (2014) 377–386

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

j ournal homepage: http://www.elsevier.com/locate/ijantimicag

Review

Alkaloids: An overview of their antibacterial, antibiotic-enhancing and antivirulence activities

a,∗ b c

T.P. Tim Cushnie , Benjamart Cushnie , Andrew J. Lamb

Faculty of Medicine, Mahasarakham University, Khamriang, Kantarawichai, Maha Sarakham 44150, Thailand

Faculty of Pharmacy, Mahasarakham University, Khamriang, Kantarawichai, Maha Sarakham 44150, Thailand

School of Pharmacy and Life Sciences, Research Institute for Health and Wellbeing, Robert Gordon University, Riverside East, Garthdee Road, Aberdeen

AB10 7GJ, UK

a r t i c l e i n f o a b s t r a c t

Article history: With reports of pandrug-resistant bacteria causing untreatable infections, the need for new antibac-

Received 16 June 2014

terial therapies is more pressing than ever. Alkaloids are a large and structurally diverse group of

Accepted 20 June 2014

compounds that have served as scaffolds for important antibacterial drugs such as metronidazole

and the quinolones. In this review, we highlight other alkaloids with development potential. Natural,

Keywords:

semisynthetic and synthetic alkaloids of all classes are considered, looking ﬁrst at those with direct

Alkaloid

antibacterial activity and those with antibiotic-enhancing activity. Potent examples include CJ-13,136, a

Antibacterial

novel actinomycete-derived quinolone alkaloid with a minimum inhibitory concentration of 0.1 ng/mL

Structure–activity

against Helicobacter pylori, and squalamine, a polyamine alkaloid from the dogﬁsh shark that renders

Mechanism of action

Synergy Gram-negative pathogens 16- to >32-fold more susceptible to ciproﬂoxacin. Where available, informa-

Antivirulence tion on toxicity, structure–activity relationships, mechanisms of action and in vivo activity is presented.

The effects of alkaloids on virulence gene regulatory systems such as quorum sensing and virulence

factors such as sortases, adhesins and secretion systems are also described. The synthetic isoquinoline

alkaloid virstatin, for example, inhibits the transcriptional regulator ToxT in Vibrio cholerae, preventing

expression of cholera toxin and ﬁmbriae and conferring in vivo protection against intestinal colonisa-

tion. The review concludes with implications and limitations of the described research and directions for

future research.

1. Introduction of unknown aetiology [5,6]. Small-molecule drugs, for the time

being, remain an essential component of infection treatment and

Antibiotic resistance continues to rise and, with the emergence prevention. Two proven strategies within this paradigm are the

of pan-resistant untreatable Enterobacteriaceae and Acinetobacter development of new drugs with direct antibacterial activity (e.g.

spp., the dawn of the much forewarned post-antibiotic era has daptomycin) and adjuncts with antibiotic-enhancing activity (e.g.

arguably broken [1]. Improved antibiotic stewardship should help tazobactam) [1]. A third, as yet clinically unproven strategy, is

reduce the rate of future losses [2], but antibiotic lifespan is limited the development of drugs that disrupt bacterial pathogenesis

even with careful use [3], so this does not negate the need for by inhibiting adhesins, autoinducers and other virulence factors

new anti-infective medications. Biologicals can reduce our depend- [7].

ence on antibiotics and the selective pressure for resistance, but Historically, natural products have been a rich source of antibac-

several limitations prevent them replacing antibacterial drugs. For terial drugs. Although the 1980s saw a decline in this type of

example, safety and efﬁcacy issues preclude vaccine use in severely research in favour of more readily manipulated synthetic com-

immunocompromised patients [4], whilst narrow-spectrum activ- pound libraries, this trend is reversing. Synthetic chemical libraries,

ity precludes monoclonal antibody and phage therapy in infections it is now recognised, tend to be limited in their structural diversity

and a poor source of antibacterial leads [8]. Other factors driving

renewed interest in natural products include the discovery of new

∗ prokaryotic and eukaryotic species in formerly unexplored envi-

Corresponding author. Tel.: +66 43 754 32240x1159.

E-mail address: t [email protected] (T.P.T. Cushnie). ronmental niches [9], technological advancements in separation,

http://dx.doi.org/10.1016/j.ijantimicag.2014.06.001

378 T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386

structure elucidation, dereplication, genome mining and combi- up to ﬁve. This nitrogen may occur in the form of a primary amine

natorial biosynthesis [10,11], and, in the case of medicinal plants (RNH2), a secondary amine (R2NH) or a tertiary amine (R3N) [19]. In

and other traditional medicines, concerns that potentially use- addition to carbon, hydrogen and nitrogen, most alkaloids contain

ful medical knowledge and materials are being lost due to urban oxygen. Alkaloids can occur as monomers or they may form dimers

expansion and species extinction [12]. Efforts to develop synthetic (also known as bisalkaloids), trimers or tetramers. Such oligomers

antibacterial drugs have not been abandoned but are now more are typically homooligomers, but heterooligomers also occur

focused on derivatization of natural molecules and synthesis of [14].

natural-product-like compounds using well-known natural prod- No single taxonomic principle exists that would allow con-

uct scaffolds [13]. sistent classiﬁcation of all alkaloids. Where possible, alkaloids

Alkaloids are a large and structurally diverse group of natural are classiﬁed according to chemical structure, biochemical ori-

products of microbial, plant and animal origin. Responsible for the gin and/or natural origin [14,18]. In terms of chemical structure,

beneﬁcial effects of traditional medicines such as cinchona bark, there are two broad divisions of alkaloids: the heterocyclic alka-

but also the harmful effects of poisons such as ergot, they have a loids (also known as typical alkaloids) that contain nitrogen in

reputation as both Nature’s curse and blessing [14]. Alkaloids have the heterocycle; and the non-heterocyclic alkaloids (also known

inspired the development of several antibacterial drugs, with syn- as atypical alkaloids or protoalkaloids) that contain nitrogen in

thesis of quinine serendipitously yielding the quinolones, structural a side chain [18]. Heterocyclic alkaloids are typically classiﬁed

alteration of azomycin yielding metronidazole, and work with the according to their ring structure (Fig. 1a), although the structural

quinoline scaffold yielding bedaquiline. In other drugs, alkaloids are complexity of these sometimes exceeds the number of subdivisions

present as scaffold substructures, e.g. linezolid and trimethoprim. [37]. Non-heterocyclic alkaloids, by comparison, are often divided

Alkaloids remain the focus of much research, their development according to biosynthetic origin. This is possible because their

as antibacterial drugs pursued within academia, industry and joint amino acid precursors remain largely intact in the alkaloid struc-

ventures [15–17]. This review seeks to integrate knowledge from ture [37]. Classiﬁcation according to natural origin is also possible,

the extensive and often widely scattered literature on antibac- since specific alkaloids are typically confined to specific sources

terial alkaloids. Naturally occurring, semisynthetic and synthetic [18].

alkaloids are all included provided they are structurally novel Individual alkaloids are assigned names in various ways, but

with chemotherapeutic or chemoprophylactic potential. Studies almost all names end with the letters ‘-ine’ [19]. Most alkaloids

with well-characterised pharmacophores already in clinical trials are named after the organism or part of the organism from which

or clinical use have been excluded, as have studies investigating they were isolated (e.g. atropine from Atropa belladonna) [18,19].

alkaloids as immunomodulators. Structural information on all the When multiple alkaloids are obtained from the same source, a pre-

described alkaloids is presented in Supplementary Table S1. ﬁx or more complicated sufﬁx is used (e.g. quinine, hydroquinine,

quinidine) or alternatively a series of letters (e.g. epicoccarine A,

epicoccarine B) [19,38]. Alkaloids may also be named after the geo-

2. Occurrence, functions, structure, classiﬁcation and graphic location of their source (e.g. tasmanine was isolated from

nomenclature a Tasmanian plant) [14], their pharmacological activity (e.g. eme-

tine induces vomiting) or, in some cases, after their discoverer (e.g.

Alkaloids are found in bacteria, fungi, plants and animals, pelletierine after Prof. Pelletier) [19].

although their distribution within each kingdom is quite limited.

They occur in ca. 300 plant families, speciﬁc compounds typically

conﬁned to certain families (e.g. hyoscyamine in Solanaceae) [18]. 3. Physiochemical, pharmacological and toxicological

Alkaloids can occur in any part of the plant, though speciﬁc com- properties

pounds may be limited to a certain part (e.g. quinine in cinchona

tree bark) [19]. In terrestrial animals, alkaloids have been reported Despite their structural diversity, alkaloids share many physical

in insects [20,21], amphibians [22,23], reptiles [24], birds [25] and chemical properties. Because they possess a nitrogen atom with

and mammals [26]. Marine animals producing alkaloids include an unshared pair of electrons, alkaloids are basic (hence their name,

sponges [27], asteroids [28], tunicates [29,30], scleractinians [31] which literally means alkali-like) [18,19]. The degree of this basicity

and the dogﬁsh shark [32]. To date, more than 18,000 alkaloids varies depending on the structure of the molecule and the loca-

have been discovered [29]. tion of other functional groups. Most alkaloids are solids, but those

Multiple roles have been attributed to alkaloids in the above that lack oxygen (e.g. coniine) are liquids. Alkaloids are insoluble

organisms, most related to self-preservation, inhibition of competi- or sparingly soluble in water, unless reacted with an acid to form a

tors, or communication. In micro-organisms, for example, alkaloids salt. Alkaloids are soluble in non-polar solvents such as chloroform,

act as feeding deterrents [33], allelochemicals, autoinducers and but their salts are not [19].

siderophores [34]. In plants, the inhibitory effects of alkaloids on Possessing a proton-accepting nitrogen atom and one or more

glycosidase and trehalose metabolism deter herbivores [35], and proton-donating amine hydrogen atoms, alkaloids readily form

the ability to quench singlet oxygen confers protection against hydrogen bonds with proteins, enzymes and receptors. This, cou-

this toxic photosynthetic byproduct [18]. Alkaloids also act as phy- pled with the frequent presence of proton-accepting and -donating

toanticipins and phytoalexins, protecting plants against infection functional groups such as phenolic hydroxyl and polycyclic moi-

[36]. In the animal kingdom, rove beetles release the surface-active eties, explains the exceptional bioactivity of the alkaloids [39].

alkaloid stenusine to ‘skim’ across water away from danger [20], Pharmacological properties include analgesic (e.g. codeine), cen-

poison dart frogs secrete batrachotoxin as a defence against snake tral nervous stimulant (e.g. brucine), central nervous depressant

predation [22], and arctiid moths use pyrrolizidine alkaloids as a (e.g. morphine), antihypotensive (e.g. ephedrine), antihyperten-

courtship pheromone [21]. In sponges, alkaloids deter feeding and sive (e.g. reserpine), antipyretic (e.g. quinine), anticholinergic (e.g.

protect against infection [27], whilst in scleractinians they act as atropine), antiemetic (e.g. scopolamine) [19], oxytocic and vaso-

allelochemicals [31]. constrictor (e.g. ergometrine) [37], antitumour (e.g. vinblastine)

Alkaloids are characterised by great structural diversity, the and antimalarial (e.g. quinine) [18] activities. These activities are

presence of a basic nitrogen atom being the only unifying feature exploited both in traditional medicine (e.g. quinine-rich cinchona

[18]. Most alkaloids possess just one nitrogen atom, but some have bark in the treatment of malaria) [37] and modern medicine

T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386 379

Fig. 1. Skeleton structures of (a) the major heterocyclic alkaloid classes possessing antibacterial activity, and important antibacterial subclasses of the (b) indole and (c)

isoquinoline alkaloids.

(e.g. vinblastine in the treatment of cancer) [18]. Other alkaloids 4. Direct antibacterial activity

have been incorporated into human culture as recreational drugs

and drugs of abuse (e.g. caffeine, nicotine, psilocybin, cocaine) 4.1. Naturally occurring alkaloids

[18,19].

Some alkaloids are highly toxic and there have been many inci- Studies describing naturally occurring antibacterial alkaloids

dents of human poisoning [18]. In studies of 350 plant-derived date back to the 1940s, but much of this early work stopped short

pyrrolizidine alkaloids, approximately one-half were hepatotoxic of determining minimum inhibitory concentrations (MICs). Sub-

and several were carcinogenic [40]. This is due to mammalian sequent research has been more thorough, and several potently

liver oxidases transforming the compounds into reactive pyrrole antibacterial alkaloid monomers (MICs ≤10 ␮g/mL) have been

structures that alkylate nucleic acids and proteins [37]. Also, the identiﬁed in the aaptamine [46], indole [47–52], indolizidine

furoquinolines are phototoxic and photomutagenic due to the furan [53], isoquinoline [54–59], piperazine [60], quinoline [61,62],

double bond reacting covalently with DNA [41]. Other examples quinolone [63], agelasine [64,65] and polyamine [32] classes. Alka-

include aconitine (cardiotoxic) [42], lycorine (enterotoxic) [43], loid dimers with similar levels of activity have been found in the

nicotine (teratogenic) [44] and strychnine (neurotoxic) [45]. Some aaptamine-indole [66], bisindole [67,68], indole-quinoline [69,70],

of the alkaloids used in medicine, at supratherapeutic doses, can pyridoacridine [71,72], bispyrrole [73–75] and pyrrole-imidazole

also be toxic. Well-known historical examples include belladonna [76] classes. A list of compounds with the lowest recorded MICs is

(containing atropine) used as a poison in Roman times, and ergot presented in Table 1. Toxicity data for these agents are limited, but

(containing ergometrine) responsible for thousands of deaths in the in vitro chelerythrine [59], hapalindole I [52] and prosopilosidine

Middle Ages [19,37]. [53] are antibacterial at concentrations that are non-haemolytic or

380 T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386

Table 1

Information on the 10 most potently antibacterial natural alkaloids as identiﬁed by PubMed and ScienceDirect searches (no date restrictions).

a a b

Alkaloid Alkaloid class (subclass) MIC assay Cell density (CFU/mL) MIC (␮g/mL) Reference

Gram-positive Gram-negative Mycobacteria

5 c

CJ-13,136 Quinolone BMAD 1 × 10 NA 0.0001 NT [63]

8 c

Ascididemin Pyridoacridine BMID 2 × 10 0.08 0.06 NT [71]

Xinghaiamine A Miscellaneous BMID 1 10 0.3–4 0.1–2 NT [77]

5 c

Eudistomin Y4 Indole (␤-carboline) BMID 5 × 10 0.8–3.1 0.4–50 NT [51]

5 c

Hapalindole I Indole MABA 1 × 10 NA NA 0.7 [52]

Squalamine Polyamine BMID 1 × 10 1–2 1 to >100 NT [32,78,97]

5 c

Prosopilosidine Indolizidine BMID 5 × 10 1.3 NT 2.5 [53]

Clausenol Indole (carbazole) AD NS 1.3–14 7–14 NT [47]

5 c

× Chelerythrine Isoquinoline (benzophenanthridine) BMID 1 10 1.5 1.5 NT [59]

d c

8-ADHN Isoquinoline NS NS 1.6 NA NT [56]

d 5

Agelasine D Agelasine MTT 1 × 10 NT NT 1.6–3.1 [65]

d 6

Sanguinarine Isoquinoline (benzophenanthridine) BMID 1 × 10 1.6–6.3 NT NT [58]

MIC, minimum inhibitory concentration; BMAD, broth macrodilution assay; NA, not active (MIC > 100 ␮g/mL); NT, not tested; BMID, broth microdilution assay; MABA,

microplate alamarBlue assay; AD, agar dilution assay; NS, not stated; MTT, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay.

Where MIC protocols were not stated or were ambiguous in the journal article, authors were contacted for further details.

MIC ranges listed are for human pathogens.

Studies that identiﬁed more than one highly active compound.

8-ADHN (8-acetonyldihydronitidine), agelasine D and sanguinarine have comparable levels of antibacterial activity, so all three have been included.

non-toxic to Vero cells, and in vivo squalamine is effective against Lastly, in the thiazole class, a 1,3,4-thiadiazole derivative (com-

Pseudomonas aeruginosa pneumonia at 0.15 mg/kg body weight, a pound 6f) has been synthesised with an MIC of 1.56 ␮g/mL against

dose 65-fold lower than known safe levels [15]. Studies with san- M. tuberculosis [88], and a rhodanine derivative (CCR-11) has been

guinarine suggest that a daily oral dose of 5 mg/kg body weight synthesised with an MIC of 1.07 ␮g/mL against Bacillus subtilis [89].

is safe in animals [79] and, used topically, this agent has proven Compound 6f has not been assessed for toxicity, but studies with

clinical safety and efﬁcacy in orthodontic patients [80]. The alkyl HeLa cells suggest that CCR-11 activity is selective.

methyl quinolone alkaloids 1-methyl-2[(Z)-7-tridecenyl]-4-(1H)-

quinolone and 1-methyl-2[(Z)-8-tridecenyl]-4-(1H)-quinolone are

also worth mentioning here. In combination, these compounds 4.3. Structure–activity relationships (SARs)

have MICs of 0.02–0.05 ␮g/mL against Helicobacter pylori and

proven in vivo efﬁcacy against H. pylori infection [81]. SARs have been investigated for various subclasses of the

indole (Fig. 1b) and isoquinoline (Fig. 1c) alkaloids. For ␤-carboline

indoles, dimerisation improves antibacterial activity [82], possibly

because the larger molecule is less susceptible to bacterial efﬂux.

4.2. Semisynthetic and synthetic alkaloids Amidation of ␤-carbolines at C-8 reduces toxicity, and if a hexamido

group is used the reduction in toxicity is accompanied by a >20-fold

Efforts to improve alkaloid activity through synthetic modiﬁca- increase in antibacterial activity [84]. Activity also increases with

tions have been successful when using indole, isoquinoline, pyrrole increasing numbers of bromine atoms [51]. For carbazole indoles,

␤

and thiazole alkaloids as scaffolds. In the indole class, a -carboline hydroxylation at C-1 and isoprenylation at C-4 both improve activ-

dimer (NCD9; Supplementary Table S1) and simple indole dimer ity, whilst methoxylation at C-1 reduces activity [49].

(5,6,6 -tribromo-1H,1 H-2,2 -biindole) have been synthesised, with For simple isoquinoline alkaloids, addition of alkyl substituents

␮ ␮

respective MICs of 0.1–4.0 g/mL [82] and 0.5 g/mL [83] against at C-1 improves antibacterial activity, but these improvements

Gram-positive pathogens. Also, a manzamine A derivative has been appear not to be selective [54]. For benzophenanthridine iso-

␮

produced (8-n-hexamidomanzamine A) with an MIC of 0.31 g/mL quinolines, a methylenedioxy functional group at C-2 and C-3 is

against Mycobacterium intracellulare [84]. Toxicity tests with CHO- important for activity [17,59]. Addition of a phenyl or biphenyl

K1 [82], HEK 293 [83] and Vero cells [84] suggest that these indoles substituent at C-1 or C-12 can improve activity, and further

have selective activity. improvements can be achieved by adding a methoxy group at C-7

In the isoquinoline class, a biphenyl-substituted sanguinar- and C-8 [17]. For protoberberine isoquinolines, a methylenedioxy

ine derivative (compound 9) has been produced with an MIC functional group at C-2 and C-3 improves activity [90], as does a

␮

of 0.5 g/mL against Staphylococcus aureus [including meticillin- phenoxyalkyl group at C-9 [91].

resistant S. aureus (MRSA)] [17]. Also, berberine has been

conjugated with a multidrug resistance pump inhibitor to cre-

ate a compound (SS14) with MICs of 1.8–3.7 ␮g/mL against a 4.4. Identiﬁcation of activity as bacteriostatic or bactericidal

range of Gram-positive pathogens [85]. Toxicity of the sanguinar-

ine derivative has not been tested [17], but the berberine derivative Antibacterial agents that kill bacteria are more versatile than

is non-toxic at >100 ␮g/mL and shows efﬁcacy in an in vivo model those that just inhibit growth as they can be used as short-

of enterococcal infection [85]. term therapies [92], against deep-seated [93,94] and immediately

In the pyrrole class, hybrids of 4,5-dibromopyrrole and 1,3,4- life-threatening infections [94], and in immunocompromised and

oxadiazole have been produced with MICs of 1.6 ␮g/mL against immunosuppressed patients [94,95]. In the absence of confounding

S. aureus and Escherichia coli and MICs of 1.5–1.6 ␮g/mL against factors, bactericidal agents are deﬁned as those causing a ≥99.9%

Mycobacterium tuberculosis [86]. Also, a hybrid of 1-methyl- decrease in bacterial viability at concentrations no more than four

4,5-dibromopyrrole and aroyl hydrazone has been synthesised times the MIC [96]. Most studies indicate that alkaloids are bacteri-

(compound 4 m) with MICs of 0.2–0.8 ␮g/mL against S. aureus cidal [62,97–100], although this can be species-dependent for some

(including MRSA) [87]. Toxicity data for these compounds are not alkaloids (e.g. chelerythrine, prosopilosidine) [53,59]. Squalamine

yet available. has been shown to be rapidly bactericidal, with MIC levels

T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386 381

Table 2

Information on the ﬁve most potently synergistic alkaloid/antibiotic combinations (natural and synthetic) as identiﬁed by PubMed and ScienceDirect searches (no date restrictions).

Alkaloid Alkaloid class Antibiotic Test bacteria Reduction in antibiotic MIC Reference

Naturally occurring alkaloids

b c

Squalamine Polyamine Ciproﬂoxacin Klebsiella pneumoniae 16- to >32-fold [110]

d c

Lysergol Ergoline Tetracycline Escherichia coli 8-fold [118]

Tetrandrine Bisisoquinoline Cefazolin MRSA 4-fold [107]

e c

Compound 8 Pyridine Ciproﬂoxacin Staphylococcus aureus 3-fold [116]

f c

Tomatidine Steroidal Gentamicin S. aureus 2- to 32-fold [108]

Semisynthetic and synthetic alkaloids

INF392 Pyrimidine Ciproﬂoxacin Bacillus subtilis 8-fold [112]

Amlodipine Piperidine Streptomycin Salmonella enterica serotype Typhimurium 6.5- to 8-fold [113]

Compound 13 Quinoline Levoﬂoxacin Pseudomonas aeruginosa 4- to 8-fold [117]

b c

GG918 Acridine-isoquinoline Norﬂoxacin S. aureus 4- to 8-fold [119]

Compound 4e Pyrrole-imidazole Oxacillin MRSA 4-fold [114]

MIC, minimum inhibitory concentration; MRSA, meticillin-resistant S. aureus.

All of the studies tested for synergy using the broth microdilution method and/or chequerboard test.

Efﬂux pump overproducing.

Studies that detected synergy with more than one antibiotic and/or against more than one species. d Multidrug-resistant.

NorA (efﬂux pump) expressing.

Both antibiotic-sensitive and multidrug-resistant strains.

Semisynthetic alkaloids that showed no improvement in activity compared with their parent structure have been excluded.

reducing the viability of Gram-positive and Gram-negative causes leakage of cytoplasmic contents [97,106]. The suscepti-

≥

pathogens by 99.99% in just 1–2 h [97]. bility of porin-negative, efﬂux-pump-overproducing bacteria to

squalamine is consistent with this hypothesis [106].

4.5. Mechanisms of action

5. Synergistic and antibiotic resistance-modulating activity

Antibacterial mechanism of action (MOA) has been investigated

for alkaloids in the indolizidine, isoquinoline, quinolone, agelasine 5.1. Naturally occurring alkaloids

and polyamine classes. In the indolizidine class, it has been pro-

posed that the alkaloids pergularinine and tylophorinidine act by Some alkaloids have been reported to increase the antibacterial

inhibiting nucleic acid synthesis, as they inhibit the enzyme dihy- activity of antibiotics, and information on the ﬁve most potent com-

drofolate reductase in cell-free assays [101]. binations is presented in Table 2. For tetrandrine and tomatidine,

In the isoquinoline class, two MOAs have been proposed. Studies this activity has been conﬁrmed as synergistic (not additive) by

with the benzophenanthridine and protoberberine isoquinolines determining fractional inhibitory concentration index (FICI) values

suggest that these act by perturbing the Z-ring and inhibiting cell [107,108]. Although the reductions in antibiotic MICs are modest

division. Supporting evidence includes demonstrations that san- compared with other natural products [109], test alkaloids can

␮

guinarine and berberine (a) bind to FtsZ [102,103], (b) inhibit FtsZ exert this effect at quite low concentrations (e.g. 0.8–1.6 g/mL

GTPase activity [103], (c) inhibit Z-ring formation [102–104] and (d) for squalamine) [110]. It is also worth noting that comparatively

induce cell elongation [102,104] without affecting DNA replication, few alkaloids have been tested to date and, based on hit ratio, it is

nucleoid segregation or membrane structure [102] and without thought many active compounds still await discovery [111].

inducing the SOS response [104]. Overexpression and underex-

pression studies also support this mechanism [104]. Researchers 5.2. Semisynthetic and synthetic alkaloids

working with the phenanthridine isoquinoline ungeremine sug-

gest that this alkaloid acts by inhibiting nucleic acid synthesis after In addition to natural alkaloids, various semisynthetic and syn-

observing inhibition of type I topoisomerases in cell-free assays thetic alkaloids have been reported to increase antibiotic activity

[105]. (Table 2). This activity has been conﬁrmed as synergistic for INF392,

Naturally occurring quinolone alkaloids lack the 3-carboxyl amlodipine and compound 4e [112–114] and occurs at quite low

␮ ␮

group that enables synthetic quinolones such as the ﬂuoro- alkaloid concentrations (e.g. 0.4 g/mL for INF392 and 2.5 g/mL

quinolones to inhibit the type II topoisomerase enzymes [34]. for compound 13). For amlodipine, this synergy has been demon-

Research with the alkyl methyl quinolones suggests that these are strated in vivo, with intraperitoneal injection of amlodipine and

respiratory inhibitors, as they reduce O2 consumption in treated streptomycin protecting mice against Salmonella enterica serotype

bacteria but do not affect H uptake [81]. Typhimurium infection [113]. A second beneﬁcial effect noted with

The agelasines are a class of alkaloids from the Agelas marine some of these compounds is that the emergence of antibiotic resis-

sponges [64,65]. Overexpression and binding afﬁnity studies with tance can be inhibited. For example, INF392 reduces the rate at

the antimycobacterial alkaloid agelasine D suggest that this exerts which ciproﬂoxacin resistance emerges by 100-fold [112].

its antibacterial effect by inhibiting enzyme BCG 3185c, a suspected

dioxygenase, thereby disrupting bacterial homeostasis [65]. 5.3. Structure–activity relationships

Lastly, studies with the polyamine alkaloid squalamine suggest

that it acts by compromising outer membrane and cytoplasmic Various alkaloid classes have been shown to increase antibi-

membrane integrity. Supporting evidence includes demonstration otic activity, including the indole [111], piperidine [111,115],

that squalamine (a) penetrates reconstituted lipopolysaccharide pyridine [116], quinoline [111,117], ergoline [118], polyamine

monolayers [106], (b) causes depolarisation of the cytoplasmic [110] and steroidal [108] classes, as well as acridine–isoquinoline

membrane [97], (c) increases bacterial staining with the cell- dimers [119], isoquinoline dimers [107], pyrrole-imidazole dimers

impermeable nucleic acid dye propidium iodide [106] and (d) [114] and pyridine–pyridine–piperidine trimers [120]. For the

382 T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386

Table 3

Alkaloids identiﬁed as quorum sensing (QS) inhibitors based on their effects upon QS-regulated virulence factors and processes and/or their interactions with speciﬁc QS

targets.

Alkaloid (alkaloid class) Virulence factors/processes inhibited QS target(s) Reference

Compound 37 (1,3,4-oxadiazole) Inhibits toxin (pyocyanin) and QS signal precursor (HHQ) production in Pseudomonas aeruginosa PqsR [127]

7-Hydroxyindole (indole) Alters virulence gene expression, inhibits toxin (pyocyanin), QS signal (PQS), biosurfactant PQS, PqsR [129]

(rhamnolipid) and siderophore (pyochelin) production, and abolishes swarming motility in

P. aeruginosa

Solenopsin A (piperidine) Inhibits virulence gene transcription, toxin (pyocyanin) and destructive enzyme (elastase B) rhl system [128]

production, and bioﬁlm formation in P. aeruginosa

Tomatidine (steroidal) Inhibits virulence gene expression and haemolytic activity in Staphylococcus aureus agr system [108]

HHQ, 2-heptyl-4-(1H)-quinolone (a precursor of PQS); PQS, Pseudomonas quinolone signal [2-heptyl-3-hydroxy-4(1H)-quinolone]; PqsR, PQS transcriptional regulator (also

known as ‘multiple virulence factor regulator’ or ‘MvfR’).

steroidal alkaloid tomatidine, activity is dependent upon the identiﬁed in the indole, 1,3,4-oxadiazole, piperidine and steroidal

spiroaminoketal moiety being in the closed conﬁguration [121]. alkaloid classes (Table 3). For the indoles, undesirable effects such

Active compounds in other alkaloid classes lack an identiﬁable as antibiotic antagonism and bioﬁlm stimulation were initially a

pharmacophore, but are often lipophilic [111], possess an aro- problem [129], but these activities have been eliminated in a new

matic ring [111,122] and have a centrally located nitrogen atom semisynthetic derivative, 7-ﬂuoroindole [130].

[111,122]. The absence of a more rigid SAR for these alkaloids may

be related to their MOA. Most are thought to effect synergy by 6.2. Inhibition of sortase

inhibiting bacterial efﬂux pumps, pumps that themselves act on

In Gram-positive bacteria, surface proteins such as adhesins,

a wide range of structurally unrelated compounds.

internalins and immune evasion proteins are attached to the

cell wall by enzymes called sortases. Recent studies using sub-

5.4. Mechanisms of action

cellular assays show that several aaptamine [131], isoquinolone

[132], pyrrolidine [133] and bisindole-imidazole [134] alkaloids

With the exception of squalamine, which enhances antibiotic

inhibit sortase A. Mass spectrometry studies indicate that, for the

activity by permeabilising cells [110], and tomatidine, whose MOA

pyrrolidine alkaloids, enzyme inhibition occurs due to covalent

is unknown [121], most alkaloids act through efﬂux pump inhibi-

modiﬁcation of the active site nucleophile Cys184 [133]. The impact

tion. This MOA was established through studies with the model

of this sortase inhibition has also been demonstrated at the cellular

efﬂux pump substrate ethidium bromide (EtBr). EtBr ﬂuoresces

level. Whole cells of S. aureus treated with sub-MIC bromodeoxy-

when bound to nucleic acid, and active alkaloids reduce the rate

topsentin [134] and isoaaptamine [131] exhibit decreased binding

at which EtBr-loaded cells lose ﬂuorescence [112,118–120]. If it

to ﬁbronectin, the host cell receptor that allows bacteria to bind to

can reach a sufﬁciently high intracellular concentration, EtBr is also

mucous membranes.

antibacterial, and active alkaloids have been shown to reduce EtBr

MICs [111,112,123]. For the ergoline alkaloid lysergol, efﬂux inhi-

6.3. Disruption of ﬁmbriae and other adhesins

bition is thought to occur due to downregulation and inhibition

of efflux pump ATPases [118]. Efflux pumps with confirmed sus-

Alkaloids can also disrupt adhesins via non-sortase-mediated

ceptibility to alkaloid inhibition include NorA [112], MexAB–OprM,

mechanisms. Sub-MIC levels of the isoquinoline berberine cause

MexCD–OprJ, MexEF–OprN [117] and ABC transporters [118].

Streptococcus pyogenes cells to release lipoteichoic acid, reducing

their ability to bind ﬁbronectin [135]. Sub-MIC levels of berber-

6. Attenuation of bacterial pathogenicity

ine and 2-pyridone alkaloids disrupt E. coli ﬁmbriae via a different

mechanism. For berberine, the MOA underlying inhibition of ﬁm-

Bacterial pathogenesis is a multistage process typically involv-

brial synthesis is not clearly established [136], but the 2-pyridones

ing bacterial attachment to host skin or mucous membranes,

disrupt the FGS [137,138] and FGL [139] assembly systems by bind-

multiplication, evasion of host defences, then toxin production

ing to periplasmic chaperones. Whole-cell studies conﬁrm that

and/or invasion and inﬂammation [94]. This process is dependent

this results in inhibition of ﬁmbria-dependent bioﬁlm formation

upon numerous virulence factors, expression of which is tightly

in E. coli [137].

regulated.

6.4. Inhibition of bacterial defences against the host immune

6.1. Disruption of virulence gene regulation

system

One mechanism by which bacteria regulate virulence is the use

Disruption of sortase-dependent immune evasion proteins (e.g.

of transcriptional regulators sensitive to environmental conditions.

protein A) is not the only mechanism by which alkaloids increase

In Vibrio cholerae, the transcriptional regulator ToxT responds to the

bacterial susceptibility to the host immune system. The thia-

presence of intestinal fatty acids and bicarbonate by activating the

zole alkaloid D157070 (a prodrug for D155931) has been shown

genes encoding cholera toxin and ﬁmbriae [124]. Recent research

to inhibit dihydrolipoamide acyltransferase, the enzyme that M.

shows that the isoquinoline alkaloid virstatin inhibits production of

tuberculosis uses to detoxify reactive nitrogen intermediates in host

both of these virulence factors, with microarray and mutant studies

macrophages. Whole-cell studies with mycobacteria conﬁrm that

identifying ToxT as the likely target [125,126]. Virstatin also has a

D157070 increases their sensitivity to nitrite and promotes killing

protective effect in vivo, inhibiting intestinal colonisation of infant

of cells in infected bone-marrow-derived mouse macrophages

mice when administered during or after V. cholerae inoculation [140].

[125].

Quorum sensing (QS) is another regulatory mechanism. Signal

6.5. Inhibition of secretion systems

molecules (autoinducers) released by bacteria bind to receptors

in neighbouring cells, triggering virulence factor expression when Bacteria use secretion systems to assemble surface structures

a critical population density is reached. QS inhibitors have been such as adhesins and to export toxins and destructive enzymes.

T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386 383

High-throughput screening has identiﬁed TTS29, a 2-imino-5- needs to be established, as some antibacterial (e.g. manzamine A

arylidene thiazolidinone that inhibits Gram-negative type II and [149]) and antibiotic-enhancing (e.g. reserpine [112,120]) alkaloids

type III secretion systems. The thiazole has broad-spectrum activ- are highly toxic to eukaryotic cells. Because some alkaloids are

ity, inhibiting secretion in multiple bacterial species, and protects immunosuppressive (e.g. gliotoxin [150]), toxicity testing should

macrophages against S. Typhimurium cytotoxicity. Based on its evaluate this parameter too.

activity proﬁle, and molecular studies to rule out interference In addition to direct antibacterial and antibiotic-enhancing

with transcription or translation, the alkaloid is thought to target activities, alkaloid inhibition of bacterial virulence has been

secretin, an outer membrane protein conserved across the type II described. For alkaloids such as berberine that exert both direct

and type III systems [141]. Monomeric [141] and dimeric [142] ana- antibacterial and antivirulence effects, the implication is that these

logues of TTS29 have been produced with enhanced solubility and will, like the QS-inhibiting macrolide antibiotics [151], exert greater

increased activity. in vivo efﬁcacy than their MIC evaluations suggest. For alkaloids

that inhibit bacterial virulence without affecting growth or viabil-

6.6. Inhibition of exotoxin-mediated effects ity, these could potentially be developed as antivirulence drugs. A

common concern with antivirulence drugs is that narrow-spectrum

Toxins are one of the main mechanisms by which bacteria cause activity could preclude empirical use. Until real-time diagnos-

disease. In vivo research with the isoquinoline berberine shows tics are more widely available, efforts should therefore focus on

that this counteracts V. cholerae cholera toxin and E. coli heat-stable alkaloids likely to have broad-spectrum activity, i.e. inhibitors of

enterotoxin. Studies were performed with the toxins not the bacte- secretion systems [141] and other virulence factors conserved

rial cells, so the protective effect cannot be related to inhibition of across bacterial species. A second concern with antivirulence drugs

virulence gene regulation, bacterial attachment or toxin secretion. is that, because they do not kill bacteria, they could not be used

Given that these enterotoxins differ in their MOA, and berberine in immunocompromised patients. It may be possible to over-

neither inhibits adenylate cyclase nor enhances intestinal absorp- come this problem by targeting virulence factors required for

tion, it is thought the alkaloid acts at a biochemical step after cyclase colonisation and maintenance of attachment (e.g. adhesins and

activation [143]. adhesin-synthesising machinery) rather than disease symptoms

(e.g. toxins).

6.7. Inhibition of destructive enzyme-mediated effects Regarding future discovery and development efforts, there are

opportunities to expedite this process. By departing from what,

Another major mechanism by which bacteria cause disease is in academia, is often a monodisciplinary approach and promot-

invasion and inﬂammation. Bacteria produce destructive proteo- ing greater collaboration between chemists, microbiologists and

lytic and glycolytic enzymes, enabling them to breach host tissue pharmacologists, it should be possible for more groups to isolate

barriers and spread to deeper tissue. Whole-cell studies show that active compounds and generate robust microbiological and toxico-

berberine inhibits bacterial hydrolysis of the connective tissue logical data. Given the risk of toxicity with alkaloids, tests for overt

component collagen [144]. It remains to be established whether toxicity should be performed at the earliest opportunity. Where

this is due to inhibition of collagenase production, secretion or cell culture is available, non-malignant cell lines should be tested.

enzymatic action, or an indirect consequence of the alkaloid’s In more resource-limited settings, tests such as the brine shrimp

antibacterial activity. lethality assay or haemolysis assay could be used. Lastly, greater

use should be made of the many new techniques and technolo-

6.8. Inhibition of biofilm formation gies available, e.g. efflux pump mutants to detect efflux-susceptible

antibacterial agents [152], synergy-directed fractionation to detect

Bioﬁlm formation protects bacteria from antibiotic therapy, natural product components that work synergistically [153],

thereby prolonging infections. Numerous alkaloids inhibit the for- medicinal chemistry techniques to conjugate efﬂux-susceptible

mation of (and/or disperse) bacterial bioﬁlms, including imidazoles antibacterials with efﬂux pump inhibitors [85], and genetic tech-

[145], isoquinolines [100], piperidines [146], pyrrolidines [147], niques [104] and reporter strains of bacteria [154] for MOA

pyrrole-imidazoles [114] and cinchona alkaloids [148]. In some elucidation.

cases, this inhibition has been attributed to direct antibacterial

activity [148], in others to QS disruption [146,147] or to unknown

causes [100,114], possibly inhibition of sortase or adhesins. Acknowledgments

7. Concluding remarks We are very grateful to those authors and editors who helped

source the more-difﬁcult-to-ﬁnd journal articles used in this

Alkaloids have a proven track record as drug scaffolds and review, in particular Profs. Miki Kuftinec, Vincent G. Kokich,

scaffold substructures in modern antibacterial chemotherapy [39]. Rob Verpoorte and Adyary Fallarero. Thanks also to Dr Stephen

This review highlights other antibacterial alkaloids with develop- MacManus for constructive comments during drafting of the

ment potential, e.g. the quinolone CJ-13,136 with MICs as low as manuscript. Our apologies to authors whose work could not be

0.1 ng/mL against H. pylori, and the polyamine alkaloid squalamine included due to space restrictions.

reducing ciproﬂoxacin MICs ≥16-fold against Klebsiella pneumo-

niae. For many of these compounds, further characterisation is In memoriam

necessary, e.g. determination of spectrum of activity, MIC90 values

(i.e. MIC required to inhibit 90% of strains of a species), FICI values, We dedicate this review to our friend and colleague, the

MOA, toxicity, SAR, solubility and stability, resistance frequency, physician, lecturer and natural products researcher Dr Yash

and serum protein binding. In terms of MOA, it has been proposed, Kumarasamy (1970–2012). His warmth and enthusiasm are

based on assays with puriﬁed enzymes, that indolizidine alkaloids greatly missed.

inhibit dihydrofolate reductase and that isoquinoline alkaloids

inhibit type I topoisomerases. Whole-cell studies (e.g. target over- Funding: No funding sources.

expression, underexpression and alteration) would help rule out Competing interests: None declared.

non-speciﬁc inhibition and conﬁrm these MOAs. Selectivity also Ethical approval: Not required.

384 T.P.T. Cushnie et al. / International Journal of Antimicrobial Agents 44 (2014) 377–386

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