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Antibiotic Adjuvants: Rescuing Antibiotics from Resistance
Review
Antibiotic Adjuvants: Rescuing Antibiotics from Resistance Gerard D. Wright1,* Rooted in the mechanism of action of antibiotics and subject to bacterial evolution, antibiotic resistance is difficult and perhaps impossible to overcome. Nevertheless, strategies can be used to minimize the emergence and impact of resistance. Antibiotic adjuvants offer one such approach. These are compounds that have little or no antibiotic activity themselves but act to block resistance or otherwise enhance antibiotic action. Antibiotic adjuvants are therefore delivered in combination with antibiotics and can be divided into two groups: Class I agents that act on the pathogen, and Class II agents that act on the host. Adjuvants offer a means to both suppress the emergence of resistance and rescue the activity of existing drugs, offering an orthogonal strategy complimentary to new antibiotic discovery
Trends Resistance has emerged to all clinically used antibiotics. It is a global health care crisis that is one of the major challenges to health care in the 21st century. There is a growing gap between the clinical need for new antibiotics and new drug discovery and development. It is increasingly challenging to find new antibiotics and to bring them to market. Preserving our existing antibiotics offers a way to mitigate the gap between the need for new drugs and the diminishing supply pipeline. This can be accomplished in several ways, including the development of antibiotic adjuvants.
The Gap between Clinical Need and New Drug Discovery Public health officials the world over are sounding the alarm on the need for new antibiotics to counter a growing host of multidrug-resistant pathogenic bacteria. There are increasing numbers of reports of extremely drug-resistant bacteria for which few or no drugs are available. The result is that some patients (frequently the most vulnerable, already beset by complicated medical histories) are at risk of effectively returning to the preantibiotic era–a time when bacterial infections we are now accustomed to treating effectively, cheaply, and without major side effects are again perilously life threatening. Even the general public is now increasingly aware of the threat of antibiotic resistance [1].
Antibiotic adjuvants are nonantibiotic compounds that enhance antibiotic activity either by blocking resistance or by boosting the host response to infection. Several are already in clinical use, specifically compounds that inhibit the b-lactamases that confer resistance to b-lactam antibiotics.
The lack of new antibiotics and the continual buildup of resistance mechanisms by pathogens is not a surprise to clinicians and researchers working in the field of infectious disease. Beginning with the discovery of penicillin by Alexander Fleming (http://www.nobelprize.org/nobel_prizes/ medicine/laureates/1945/fleming-lecture.pdf), they have long highlighted the importance of resistance evolution and called for new incentives to spur antibiotic discovery and development in parallel with research into curtailing resistance. Governments, not-for-profit, and public health organizations have very recently answered with strategic plans to address resistance and boost investment in antibiotic development. These are positive additions, but the pharmaceutical industry, which has over the past two decades systematically dismantled antibiotic-discovery programs and shed expertise in antibiotic drug development, have yet to return to the field in earnest. It is therefore quite conceivable that we will not see any new antibiotics coming to market for some time. 1
Such a scenario is completely possible as there are few truly novel candidate antibiotics in clinical development. A recent review of new antibiotics for Gram-negative pathogens, an especially urgent clinical priority, identifies 17 antibiotics in either Phase 1, 2, or 3 clinical trials, only two of which are novel chemical classes with new mechanisms: brilacidin, a mimic of hostdefense peptides, and ACHN-975, an inhibitor of lipopolysaccharide biosynthesis [2]. The paucity of truly novel antibiotic chemical scaffolds reflects both the scientific and commercial
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http://dx.doi.org/10.1016/j.tim.2016.06.009
Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
*Correspondence: [email protected] (G.D. Wright).
challenges faced in new drug discovery that are unique to this therapeutic area. On the commercial side, antibiotics are expected to be cheap medicines that are given for a short period of time. The complexities of return on investment for these life-saving drugs [3,4], along with challenges in performing clinical trials (for example, demonstrating efficacy vs. relatively rare resistant organisms [5]), have conspired over the past two decades to de-incentivize new antibiotic drug development. The scientific difficulties inherent in this therapeutic area are also unique [6]. For example, the chemical matter that dominates modern drug discovery in the pharmaceutical industry is generally tailored for activity on human tissues and cells [7]. Bacteria are completely different in terms of the physical chemical properties of compounds required to penetrate cells [8]; therefore, on-target activity and potency in biochemical assays often is not replicated in studies of whole cells. In retrospective analyses, both GlaxoSmithKline [9] and AstraZeneca [10] describe decades of sustained effort that resulted in no new drug leads. We are well within the resistance era, where new drugs are rare and resistance increases unabated [11]. The new reality that we must face is that the scientific, commercial, and regulatory conditions are not presently aligned for the discovery of new drug classes. In fact they may never be as perfectly positioned as they were during the Golden Age of antibiotic drug discovery in the decade that followed the Second World War [11]. As a result, while we must never abandon the search for new antibiotic drugs, we must also steadfastly focus on preserving our existing drugs. This can be accomplished in part with judicious management of current drugs [12]. For example, we can ensure that antibiotics are used exclusively for the treatment of bacterial infection, and work toward a ‘right drug for the right bug’ approach–for example, through innovation in rapid and reliable point-of-care diagnostics, currently unavailable to physicians who, more often, rely on empiric prescription [4,13]. Continuing and enhanced education of clinicians, farmers, and the public of the dangers of inappropriate antibiotic use is also vital. These stewardship strategies must be vigorously explored if we are to slow the progression toward a postantibiotic age.
Glossary Antibiotic adjuvant: nonantibiotic molecule that potentiates the activity of an antibiotic. Class I adjuvants: compounds that block antibiotic resistance. Class I.A adjuvants block active resistance, that is, specific inactivating enzymes, bypass mechanisms etc., Class I.B adjuvants overcome metabolic or physiologic barriers to antibiotic activity, e.g., facilitate membrane penetration, block biofilms, etc. Class II adjuvants: compounds that enhance antibiotic action by interacting with host defense mechanisms. Efflux pumps: proteins and protein complexes that actively (i.e., use energy) move antibiotics out of the cell against a concentration gradient.
Antibiotic Adjuvants A complimentary strategy to protect our existing drugs is through the use of antibiotic adjuvants (see Glossary), compounds that enhance the activity of current drugs and can minimize, and even directly block, resistance [14–16]. Adjuvants are delivered together with antibiotics and therefore are combination drugs (Figure 1). The concept of antibiotic adjuvants borrows from the success of antibiotic combinations (antibiotic A + antibiotic B) in the clinic [17,18]. These have been used for decades to achieve synergy, cover microbial spectrum, and suppress resistance [19]. Well known combinations include aminoglycoside + penicillin combinations (e.g., gentamicin + ampicillin) for treatment of enterococcal infections, trimethoprim + sulfamethoxazole that has efficacy versus a number of pathogens, and multidrug therapy for treatment of tuberculosis (Box 1). Such combinations have found particular use because synergistic interactions are achieved through a variety of mechanisms, including targeting of distinct steps in common or linked biochemical pathways, enhancement of uptake or suppression of efflux. The result is that efficacy is greater than the sum of the individual agents (synergy), generally resulting in faster kill times or enhanced killing, thereby limiting the opportunity for resistant organisms to emerge. The molecular basis of antibiotic synergy underscores the importance of understanding the mechanisms of antibiotic action, including primary and secondary targets in a variety of pathogens (not just model organisms), data that are generally poorly reported, making rational selection of antibiotic pairs difficult at present; however, systems biology approaches can be applied to this problem with success [20]. Nevertheless, in the absence of mechanistic data, machine-learning strategies have recently been used to predict the efficacy of combinations in the antifungal realm and to offer an opportunity to identify new antibacterial combinations as well [21].
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Examples
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H OH
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Clavulanic acid
O
Cl
N
Loperamide
OH
H
H O
Macrophage
O
Streptazolin
Figure 1. Antibiotic Adjuvants Can Be Divided into Two Classes. When given with an antibiotic (A), Class I adjuvants inhibit active or intrinsic (passive) antibiotic resistance in bacteria, while Class II adjuvants enhance the host (H) ability to kill bacteria. Class I adjuvants can be further subdivided into Class I.A that inhibit (I) active resistance mechanism, for example, inactivating enzymes, bypass mechanisms, efflux, that are generally imported by horizontal gene transfer, and Class I.B adjuvants are nonantibiotics (N) that block passive or intrinsic mechanisms including permeability barriers such as the outer membrane in Gram-negative bacteria, biofilms, etc.
In contrast to antibiotic combinations, antibiotic adjuvants [15,16,18,22] show little or no antimicrobial activity alone. Instead, when combined with drugs, they enhance antibiotic activity under specific conditions. It is useful to classify antibiotic adjuvants into two general classes based on target profile: Class I adjuvants that work with antibiotics on bacterial targets, and Class II adjuvants that enhance antibiotic activity in the host (Figure 1). Class I adjuvants can be further differentiated based on their mechanisms. Class I.A compounds directly inhibit antibiotic resistance (inactivating enzymes, efflux pump systems, or alternate targets) and are the only adjuvants in current clinical use. Class I.B adjuvants enhance antibiotic activity by circumventing intrinsic resistance mechanisms including metabolic pathways or physiology other than direct inhibition of specific resistance elements. By contrast, Class II adjuvants do not directly impact bacteria but rather operate on host properties to potentiate antibiotic action. Examples of Class I. B and Class II adjuvants have yet to be approved by regulatory agencies as formulated drug combinations with antibiotics, but are being explored in preclinical models.
Mechanisms of Antibiotic Resistance Resistance to antibiotics occurs through a variety of molecular mechanisms, including decreased drug permeability, active efflux, alteration or bypass of the drug target, production of antibiotic-modifying enzymes, and physiological states such as biofilms that are less susceptible to antibiotic activity (Figure 2). All of these mechanisms are susceptible to inhibition by small molecules and thus are potential targets for antibiotic adjuvants. For the purposes of targeting resistance with adjuvants, it is useful to classify resistance as either active, that is, mechanisms that have evolved specifically to detoxify specific antibiotics, or passive, mechanisms that are intrinsic to specific bacteria that have the effect of resistance but are not necessarily directly targeted to an individual antibiotic. Examples of active resistance include genes acquired by horizontal and vertical transfer and the upregulation of ‘silent’ chromosomal elements that can confer drug resistance to otherwise sensitive bacteria. These include most of
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Box 1. Antibiotic Combinations and Measurement of Synergy Combining antibiotics is a strategy often used by clinicians. The objective is to ensure coverage of potential bacterial pathogens and/or resistance profiles during empiric therapy, suppress the emergence of resistance, avoid toxicity (by using less drug), or to achieve synergy. The latter is especially useful in increasing rates of cell killing and managing difficult-to-treat infectious organisms. Multidrug therapy is common in the treatment of tuberculosis for example. The causative agent, Mycobacterium tuberculosis, is a slow-growing organism that is generally found as an intracellular pathogen. Achieving adequate antibiotic concentrations and maintaining these over a sufficient period of time to ensure cell death is very difficult with only one antibiotic. Instead, a combination of three or more drugs given simultaneously is standard practice. The molecular mechanisms of synergy in antibiotic combinations are similar to those in antibiotic adjuvant pairs. They include targeting different critical but nonredundant steps in metabolic pathways (e.g., trimethoprim + sulfamethoxazole that hit distinct steps in folate metabolism), enhanced uptake of drugs (e.g., penicillin + aminoglycoside combinations in enterococci), and inhibition of redundant enzymes (e.g., targeting of multiple penicillin-binding proteins by b-lactam antibiotics). Measuring synergy in both antibiotic pairs and with antibiotic adjuvants is easily accomplished using simple plate assays, where compounds are applied on separate paper disks and allowed to diffuse on agar plates inoculated with target bacteria. Synergy is indicated with enhanced killing at the interface of the diffusion zones. Quantitative measurement of synergy can be measured using the simple checkerboard strategy, where concentrations of both agents are systematically diluted to identify concentrations of both drugs that achieve the most potent interaction. A simple mathematical device, the fraction inhibitory concentration index (FICI) between two compounds, A and B, is calculated from the following formula: FICI
¼
MICðA in combination with BÞ MICðA aloneÞ
þ
MICðB in combination with AÞ MICðB aloneÞ
(1)
In principle, any value of FICI below 1 indicates interaction that is better than additive (i.e., synergistic); however, given the accepted one dilution error of MIC determinations, compounds are reported as synergistic when FIC <0.5, that is, a fourfold decrease in MIC. In the case of adjuvant molecules that do not show an MIC, it is common and useful to use the highest concentration tested and indicate that the FICI is less than the value obtained. Measuring FICI in cases where adjuvants have no measurable MIC poses a challenge for the quantification of adjuvant potency. By analogy to the 0.5 FICI value to define synergy, the concentration of the adjuvant that reduces MIC fourfold is a reasonable marker of potency. More relevant is the concentration of adjuvant that lowers the antibiotic MIC in resistant bacteria to equal to, or less than, the breakpoint concentration, that is, the concentration of antibiotic that defines whether a strain is resistant or sensitive. The Rescue Concentration (RC) is therefore defined as the concentration of adjuvant required to lower the effective concentration of the antibiotic to less than, or equal to, the breakpoint.
the commonly known resistance elements such as detoxifying enzymes (b-lactamases, aminoglycoside acetyltransferases and kinases, etc.), bypass and target alteration mechanisms (mec genes in Staphylococcus aureus, van genes in enterococci), and some efflux systems (tetA, qac, etc.). By contrast, passive resistance occurs through nonspecific mechanisms where the antibiotic target is nevertheless sensitive to the antibiotic. These include the broad-spectrum efflux systems found in most bacteria (e.g., acrAB/tolC in Escherichia coli), mutations in porins (e.g., carbapenem resistance through mutation of oprD in Pseudomonas aeruginosa), and changes in physiology such as growth in biofilms, where antibiotic susceptibility is greatly decreased. The differences between active and passive resistance are not always simple to define, for example, upregulation of intrinsic efflux systems, but the structure is useful when considering Class I adjuvants and their targets
Class I.A Antibiotic Adjuvants: Inhibitors of Active Resistance The most clinically successful adjuvants are the inhibitors of b-lactamases, enzymes that hydrolytically inactivate penicillin, cephalosporin, and carbapenem drugs by a ring-opening mechanism (Figure 3). b-Lactamases are widespread in bacterial pathogens, and are often acquired through horizontal gene transfer; they represent the most important antibiotic-resistance challenge today given the fact that these are the most widely used antibiotics in the clinic. b-Lactamases operate by one of two molecular mechanisms: enzymes that use an active site Ser residue to covalently capture the antibiotic then release the inactive compound after
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Modifying Enzymes
Efflux
Anbioc Physiology
Permeability
Target Bypass
Figure 2. Mechanisms of Resistance to Antibiotics. These include inactivating enzymes, efflux systems, decreased permeability, physiology such as biofilm formation, and target bypass or alteration.
hydrolysis (Figure 3, path i), or enzymes that use active-site Zn2+ atoms to activate a water molecule that is positioned for hydrolysis of the antibiotic. Ser-b-lactamases are further arranged in three types: A, C, and D based on protein fold and substrate specificity. Regardless of mechanism, the end result is the same, with hydrolytic opening of the b-lactam ring that is essential for antibiotic action. In the 1970s, clavulanic acid (Figure 3), a b-lactam-containing natural product, was isolated from a strain of Streptomyces clavuligerus that also produced a b-lactam antibiotic [23]. Clavulanic acid has poor antibiotic activity, but it does show potent and irreversible inactivation of Ser-b-lactamases [24]. This discovery led to the first antibiotic/adjuvant pair to be successfully approved for clinical use. Pairing of clavulanic acid with amoxicillin generated Augmentin, a combination drug that has been in clinical use for over 30 years. The success of clavulanic acid spurred efforts to develop other b-lactamase inhibitors, including the b-lactam sulfones tazobactam and sulbactam that are paired with penicillins, respectively piperacillin and ampicillin. More recently, a new class of b-lactamase inhibitor, the diazabicyclooctanes (DABCOs) was introduced into clinical practice. The DABCO avibactam, when paired with the cephalosporin ceftazidime, forms the drug Avycaz, approved for clinical use by the FDA in 2015. Clavulanic acid is predominantly restricted to type A Ser-b-lactamases, and after three decades of clinical use many resistant alleles are circulating in pathogens. The emergence of extended-spectrum b-lactamases (ESBLs) in pathogens over the past several years has further eroded the efficacy of clavulanic acid. The penicillanic acid sulfones, such as tazobactam, showed efficacy toward some of the common ESBLs in the 1990s, but have limited effectiveness with type A carbapenemases, the increasingly common type C b-lactamases such as plasmid-born AmpC, and the oxacillinases (type D). Avibactam on the other hand does show efficacy vs. type A ESBLs such as CTX-M, AmpCs, and type A carbapenemases such as Klebsiella pneumoniae carbapenemase (KPC) [25,26]. This strategy–to block Ser b-lactamases–continues to attract innovation and imitation. For example, combinations of avibactam with ceftaroline and aztreonam are in late-stage clinical trials, and the DABCO relebactam paired with imipenem/cilastatin is also being explored. Zerbaxa (ceftolozane/tazobactam), a combination
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General mechanism of Ser (i) and metallo- (ii) β-lactamases H
R
S
(i) O
N O
H
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HN O
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O HO
Ser
O–
Ser
(i) H
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HO H O– Zn2+
Zn2+
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Inhibitors of Ser and metallo-β-lactamases HO O S
H OH
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Clavulanic Acid
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NH2
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O
S O N O N O
O
S
O O B O HO
Vaborbactam
HO OH
NH2
N H
O H N O
OH O OH
Aspergillomarasmine A
Figure 3. b-Lactamases and Inhibitors. Ser-b-lactamases (path i) are the most common in the clinic and operate through the formation of a covalent complex with the b-lactam antibiotic. The only antibiotic adjuvants in clinical use are inhibitors of these enzymes. Metallo b-lactamases (path ii) cleave the b-lactam ring of the antibiotics using a Zn-activated water molecule. No antibiotic adjuvants are yet available for these enzymes, though aspergillomarasmine A is undergoing preclinical testing.
that is especially effective vs. Pseudomonas, was approved by the FDA in 2014. Carbavance, a combination of the first in class boronic acid vaborbactam (Figure 3) with meropenem, recently received fast-track status from the FDA. The Zn-dependent metallo (type B) b-lactamases, which efficiently confer resistance to all penicillins, cephalosporins, and carbapenems, are not inhibited by the b-lactamase inhibitors in current clinical use. Historically, metallo-b-lactamases have not been a pressing clinical problem as pathogens harboring such enzymes, while alarming, have been relatively rare. However, the incidence of pathogens expressing metallo-b-lactamases is increasing globally, and the emergence of NDM-1 in 2010, which is now broadly distributed in Enterobacteriaceae, Pseudomonas, and Acinetobacter strains, is a growing concern [27]. The fact that ndm alleles are invariantly associated with multiple resistance elements spanning most (sometimes all)
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available alternative antibiotics, including aminoglycosides and recently colistin, is generating concern for the emergence of pan-drug-resistant Gram-negative bacterial pathogens. Few inhibitors of metallo-b-lactamases that are effective in animal models of infection are known. Aspergillomarasmine A (Figure 3), a fungal natural product discovered in a cell-based screen for NDM-1 inhibitors, in combination with meropenem, does show efficacy in murine models of infection [28]. The compound rescues the antibiotic activity of meropenem in pathogens expressing NDM-1 and VIM metallo-b-lactamases and operates by sequestering a Zn2+ ion essential for catalytic activity and raises the possibility of clinical use as a new Class I.A antibiotic adjuvant. The strategy of directly targeting antibiotic-resistance mechanisms with adjuvants is applicable to other resistance mechanisms and antibiotics, but thus far examples of clinical use are limited to the b-lactamases. Aminoglycoside antibiotics are bactericidal drugs that target the ribosome; they are highly effective against both Gram-negative and Gram-positive pathogens. Compounds such as gentamicin, tobramycin, and amikacin continue to find clinical use despite widespread and diverse resistance. Prevalent mechanisms include inactivating enzymes (kinases, acetyltransferases, and adenylyltransferases) and ribosome methylation [29]. Inhibitors of aminoglycoside kinases (APHs) have been reported that inhibit the enzymes and rescue antibiotic activity in resistant cells. The similarity of APHs to eukaryotic protein kinases in both structure and mechanism (despite an absence of protein sequence identity) inspired a survey of protein kinase inhibitors as potential inhibitors of APHs [30]. Several candidates were identified, but one scaffold in particular, the pyrazolopyrimidines that are human Src and PI3 kinases, inhibit and bind to APH(30 )-I (a dominant resistance mechanism in Gram-negative pathogens) in a fashion that is distinct from the binding mode in human kinases, offering a potential route for derivatives that selectively inhibit APH over human targets [31]. Other examples of blocking aminoglycoside resistance with antibiotic adjuvants include tropolones that inhibit aminoglycoside adenlylytransferases [32,33] and cationic peptides that target both APHs and acetyltansferases [34].
Class I.B Antibiotic Adjuvants: Inhibitors of Passive Resistance Bacteria can be insensitive to antibiotics because of intrinsic genetic network organization, physiology, or through the presence of nonspecific evasion mechanisms such as broad specificity efflux systems. All of these are potential targets for adjuvants. While the strategy to identify adjuvants for active resistance is relatively straightforward since the resistance targets are commonly known–for example, a b-lactamase that can be screened for inhibition using purified enzyme–adjuvants for countering passive resistance generally require cell-based screens for antibiotic potentiation followed by elucidation of mechanism (e.g., a screen for adjuvants of the semi-synthetic tetracycline antibiotic minocycline in P. aeruginosa identified several candidates, including loperamide (Imodium) [35]). Additional experiments revealed that the mode of action of loperamide, which has no antibiotic activity, was to decrease the electrical component of the proton motive force in Gram-negative bacteria. To counter this effect and maintain ATP synthesis levels, the bacteria increase the pH gradient across the inner membrane. The increase in DpH, in turn, increases uptake of tetracycline antibiotics, thereby overcoming intrinsic resistance. This observation inspired a screen for additional compounds that perturb the proton motive force in bacteria, identifying several adjuvant molecules [36]. This approach of phenotypic whole-cell screening for enhancement of antibiotic activity has revealed several adjuvants, including inhibitors of wall teichoic acid biosynthesis in methicillinresistant S. aureus (MRSA) [37–39], compounds that perturb cell shape [40], murgocil (an inhibitor of the peptidoglycan biosynthesis enzyme MurG) [20], and inhibitors of efflux systems [41]. Melander has demonstrated, through whole-cell phenotypic assays, that that 2-aminoimidazole derivatives can suppress b-lactamase activity in K. pneumoniae [42], MRSA [43], and multidrug-resistant Gram negative pathogens [44] through as yet unknown mechanisms.
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Inhibition of efflux offers a powerful strategy to enhance the activity of antibiotics. The most common antibiotic-efflux systems in bacterial pathogens are the major facilitator superfamily (MFS) and the tripartite RND systems found in many Gram-negative bacteria. These generally have broad specificities that enable active efflux of many antibiotic classes [45]. PAbN was one of the first inhibitors of RND efflux systems active in a variety of bacteria, including P. aeruginosa, which has a number of redundant efflux systems [46]. As a potent inhibitor of efflux, PAbN enhances the antibiotic activity of a number of antibiotics from different structural classes, demonstrating that such inhibitors can act as broad-spectrum adjuvants. Furthermore, combining PAbN with the fluoroquinolone antibiotic levofloxacin decreased the emergence of resistance by more than four orders of magnitude, demonstrating the value of adjuvants in suppressing the emergence of resistance [46]. Challenges with toxicity of PAbN and other efflux inhibitors have thus far prevented their use in the clinic; however, the approach is sound and it deserves increased attention. Most bacteria can readily form biofilms, and in this physiological form they are highly tolerant to antibiotics. Since biofilms are a significant problem in the clinic (e.g., indwelling medical devices, epithelial linings, etc.) compounds that enhance antibiotic activity toward biofilms have great promise as adjuvant compounds. Adjuvant molecules have the potential to act at several points in biofilm development, including prevention of biofilm formation [47–49], enhancement of antibiotic activity in mature biofilms [50], and dispersal of biofilms releasing planktonic cells that are sensitive to drugs [49,51,52].These examples reveal the depth and breadth of adjuvant targets in bacterial cells uncovered in small-molecule screens. This is supported by genetic studies of chemical synthetic lethality in E. coli. Miller and coworkers have systematically interrogated the Keio collection of E. coli BW25113 and uncovered dozens of non-obvious gene deletions that enhanced the activity of antibiotics [53]. These nonessential genes are obvious targets for antibiotic adjuvants. In particular, 61 gene deletion strains showed increased sensitivity to eight or more antibiotics. Recently, this approach has been significantly expanded and improved by Brown and colleagues using a high-throughput quantitative growth kinetics platform that identifies both subtle and strong antibiotic–genetic interactions that expand chemical genetic space by 50– 200% depending on the antibiotic interrogated [54]. This work is identifying a very rich adjuvant target landscape, and campaigns that systematically explore the drugability of these targets could prove highly effective in identifying Class I.B antibiotic adjuvants.
Class II Adjuvants: Targeting Host Defense Mechanisms Enhancing host defense mechanisms offers an alternative set of targets for antibiotic adjuvants. Immunomodulatory peptides such as LL-37 have long been known to enhance antimicrobial activity of the innate immune system, and several peptides–both with and without direct antimicrobial activity–have been in, or are in, clinical trials (reviewed in [55]). These can synergize with antibiotics as Class I adjuvants but also have the potential to act through host defense systems as Class II adjuvants [56]. Given the tremendous depth of knowledge and effort in the immunomodulatory peptide field, these likely offer the greatest potential for adjuvant-antibiotic therapy target at the host in the short term. Strategies to explore small molecule enhancement of antibiotic action in the host are worthy of exploration. For example, a screen of microbial natural product extracts for enhancers of macrophage killing activity vs. Streptococcus mutans identified the neutral compound streptazolin [57]. Mechanistic analysis revealed that streptazolin stimulates macrophage activity through the phosphoinositide 3-kinase pathway resulting in upregulation of nuclear factorkB (NF-kB). Such a screening strategy is promising as a means to identify additional Class II antibiotic adjuvants. The caveat here is the need for balance between immune modulation to increase antibiotic activity vs. the pathogen, while sparing the host the deleterious effects of overactivation of the immune system.
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Concluding Remarks
Outstanding Questions
Antibiotic resistance is one of the most pressing issues facing medicine today. New drugs candidates are increasingly difficult to identify and bring to market. As a result there is a growing gap between clinical need and drug innovation. Furthermore, the history of antibiotic discovery and use predicts that, even if we had access to a rich pipeline of drug candidates similar to those used in the past, that is, broad-spectrum single agents, resistance will continue to be a significant challenge. Antibiotic adjuvants offer an orthogonal and complementary strategy to new antibiotic discovery (see Outstanding Questions). These compounds can enhance and preserve the activity of our existing drug arsenal. The efficacy of b-lactamase inhibitors in the clinic over the past decades is proof that the concept is robust and valuable. Furthermore, by directly targeting resistance or otherwise enhancing antibiotic activity, the impact and emergence of resistance can be minimized (though never eliminated). In this new era of antibiotic resistance, we must continue to explore new strategies to preserve our antibiotic drugs and the adjuvant concept is one contribution that has great promise.
Can non-b-lactamase Class I.A adjuvants find use in the clinic? What molecular epidemiological studies are necessary to provide a suitable value proposition?
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13. Zumla, A. et al. (2014) Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections–needs, advances, and future prospects. Lancet Infect. Dis. 14, 1123–1135
32. Allen, N.E. et al. (1982) 7-Hydroxytropolone: an inhibitor of aminoglycoside-2“-O-adenylyltransferase. Antimicrob. Agents Chemother. 22, 824–831
14. Bernal, P. et al. (2013) Antibiotic adjuvants: identification and clinical use. Microb. Biotechnol. 6, 445–449
33. Hirsch, D.R. et al. (2014) Inhibition of the ANT(2”)-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic alpha-hydroxytropolones. Bioorg. Med. Chem. Lett. 24, 4943–4947
15. Gill, E.E. et al. (2015) Antibiotic adjuvants: diverse strategies for controlling drug-resistant pathogens. Chem. Biol. Drug Des. 85, 56–78 16. Kalan, L. and Wright, G.D. (2011) Antibiotic adjuvants: multicomponent anti-infective strategies. Expert. Rev. Mol. Med. 13, e5 17. Drusano, G.L. et al. (2015) Suppression of emergence of resistance in pathogenic bacteria: keeping our powder dry, Part 2. Antimicrob. Agents Chemother. 60, 1194–1201 18. Worthington, R.J. and Melander, C. (2013) Combination approaches to combat multidrug-resistant bacteria. Trends Biotechnol. 31, 177–184 19. Eliopoulos, G.M. and Eliopoulos, C.T. (1988) Antibiotic combinations: should they be tested? Clin. Microbiol. Rev. 1, 139–156
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34. Boehr, D.D. et al. (2003) Broad-spectrum peptide inhibitors of aminoglycoside antibiotic resistance enzymes. Chem. Biol. 10, 189–196 35. Ejim, L. et al. (2011) Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat. Chem. Biol. 7, 348–350 36. Farha, M.A. et al. (2013) Collapsing the proton motive force to identify synergistic combinations against Staphylococcus aureus. Chem. Biol. 20, 1168–1178 37. Farha, M.A. et al. (2013) Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to beta-lactams. ACS Chem. Biol. 8, 226–233
Can we discover effective and safe efflux pump inhibitors? What about biofilm breakers? What role can Class I.B adjuvants have in preserving legacy antibiotics and can chemical synthetic lethality studies offer an exploitable target-based pathway to such combinations? Can combinations of immunomodulatory compounds and antibiotics become commonplace in the treatment of infection? What are the barriers to such approaches?
38. Lee, S.H. et al. (2016) TarO-specific inhibitors of wall teichoic acid biosynthesis restore beta-lactam efficacy against methicillin-resistant staphylococci. Sci. Transl. Med. 8, 329ra332
48. Wenderska, I.B. et al. (2011) Palmitoyl-DL-carnitine is a multitarget inhibitor of Pseudomonas aeruginosa biofilm development. Chembiochem. 12, 2759–2766
39. Wang, H. et al. (2013) Discovery of wall teichoic acid inhibitors as potential anti-MRSA beta-lactam combination agents. Chem. Biol. 20, 272–284
49. Reffuveille, F. et al. (2014) A broad-spectrum antibiofilm peptide enhances antibiotic action against bacterial biofilms. Antimicrob. Agents Chemother. 58, 5363–5371
40. Taylor, P.L. et al. (2012) A forward chemical screen identifies antibiotic adjuvants in Escherichia coli. ACS Chem. Biol. 7, 1547–1555
50. Brackman, G. et al. (2016) The quorum sensing inhibitor hamamelitannin increases antibiotic susceptibility of Staphylococcus aureus biofilms by affecting peptidoglycan biosynthesis and eDNA release. Sci. Rep. 6, 20321
41. Cox, G. et al. (2014) An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria. J. Antimicrob. Chemother. 69, 1844–1855 42. Worthington, R.J. et al. (2012) Small molecule suppression of carbapenem resistance in NDM-1 producing Klebsiella pneumoniae. ACS Med. Chem. Lett. 3, 357–361 43. Harris, T.L. et al. (2012) Potent small-molecule suppression of oxacillin resistance in methicillin-resistant Staphylococcus aureus. Angew Chem. Int. Ed. Engl. 51, 11254–11257 44. Brackett, C.M. et al. (2014) Small-molecule suppression of betalactam resistance in multidrug-resistant gram-negative pathogens. J. Med. Chem. 57, 7450–7458 45. Li, X.Z. et al. (2015) The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 28, 337–418 46. Lomovskaya, O. et al. (2001) Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob. Agents Chemother. 45, 105–116 47. Nguyen, U.T. et al. (2012) Small-molecule modulators of Listeria monocytogenes biofilm development. Appl. Environ. Microbiol. 78, 1454–1465
51. Rabin, N. et al. (2015) Agents that inhibit bacterial biofilm formation. Future Med. Chem. 7, 647–671 52. Ribeiro, S.M. et al. (2016) New frontiers for anti-biofilm drug development. Pharmacol. Ther. 160, 133–144 53. Liu, A. et al. (2010) Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code. Antimicrob. Agents Chemother. 54, 1393– 1403 54. French, S. et al. (2016) A robust platform for chemical genomics in bacterial systems. Mol. Biol. Cell 27, 1015–1025 55. Hancock, R.E. et al. (2012) Modulating immunity as a therapy for bacterial infections. Nat Rev Microbiol 10, 243–254 56. Cassone, M. and Otvos, L., Jr (2010) Synergy among antibacterial peptides and between peptides and small-molecule antibiotics. Expert Rev. Anti. Infect. Ther. 8, 703–716 57. Perry, J.A. et al. (2015) A macrophage-stimulating compound from a screen of microbial natural products. J. Antibiot. (Tokyo) 68, 40–46
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Antibiotic Adjuvants: Rescuing Antibiotics from Resistance Gerard D. Wright1,* Rooted in the mechanism of action of antibiotics and subject to bacterial evolution, antibiotic resistance is difficult and perhaps impossible to overcome. Nevertheless, strategies can be used to minimize the emergence and impact of resistance. Antibiotic adjuvants offer one such approach. These are compounds that have little or no antibiotic activity themselves but act to block resistance or otherwise enhance antibiotic action. Antibiotic adjuvants are therefore delivered in combination with antibiotics and can be divided into two groups: Class I agents that act on the pathogen, and Class II agents that act on the host. Adjuvants offer a means to both suppress the emergence of resistance and rescue the activity of existing drugs, offering an orthogonal strategy complimentary to new antibiotic discovery
Trends Resistance has emerged to all clinically used antibiotics. It is a global health care crisis that is one of the major challenges to health care in the 21st century. There is a growing gap between the clinical need for new antibiotics and new drug discovery and development. It is increasingly challenging to find new antibiotics and to bring them to market. Preserving our existing antibiotics offers a way to mitigate the gap between the need for new drugs and the diminishing supply pipeline. This can be accomplished in several ways, including the development of antibiotic adjuvants.
The Gap between Clinical Need and New Drug Discovery Public health officials the world over are sounding the alarm on the need for new antibiotics to counter a growing host of multidrug-resistant pathogenic bacteria. There are increasing numbers of reports of extremely drug-resistant bacteria for which few or no drugs are available. The result is that some patients (frequently the most vulnerable, already beset by complicated medical histories) are at risk of effectively returning to the preantibiotic era–a time when bacterial infections we are now accustomed to treating effectively, cheaply, and without major side effects are again perilously life threatening. Even the general public is now increasingly aware of the threat of antibiotic resistance [1].
Antibiotic adjuvants are nonantibiotic compounds that enhance antibiotic activity either by blocking resistance or by boosting the host response to infection. Several are already in clinical use, specifically compounds that inhibit the b-lactamases that confer resistance to b-lactam antibiotics.
The lack of new antibiotics and the continual buildup of resistance mechanisms by pathogens is not a surprise to clinicians and researchers working in the field of infectious disease. Beginning with the discovery of penicillin by Alexander Fleming (http://www.nobelprize.org/nobel_prizes/ medicine/laureates/1945/fleming-lecture.pdf), they have long highlighted the importance of resistance evolution and called for new incentives to spur antibiotic discovery and development in parallel with research into curtailing resistance. Governments, not-for-profit, and public health organizations have very recently answered with strategic plans to address resistance and boost investment in antibiotic development. These are positive additions, but the pharmaceutical industry, which has over the past two decades systematically dismantled antibiotic-discovery programs and shed expertise in antibiotic drug development, have yet to return to the field in earnest. It is therefore quite conceivable that we will not see any new antibiotics coming to market for some time. 1
Such a scenario is completely possible as there are few truly novel candidate antibiotics in clinical development. A recent review of new antibiotics for Gram-negative pathogens, an especially urgent clinical priority, identifies 17 antibiotics in either Phase 1, 2, or 3 clinical trials, only two of which are novel chemical classes with new mechanisms: brilacidin, a mimic of hostdefense peptides, and ACHN-975, an inhibitor of lipopolysaccharide biosynthesis [2]. The paucity of truly novel antibiotic chemical scaffolds reflects both the scientific and commercial
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http://dx.doi.org/10.1016/j.tim.2016.06.009
Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
*Correspondence: [email protected] (G.D. Wright).
challenges faced in new drug discovery that are unique to this therapeutic area. On the commercial side, antibiotics are expected to be cheap medicines that are given for a short period of time. The complexities of return on investment for these life-saving drugs [3,4], along with challenges in performing clinical trials (for example, demonstrating efficacy vs. relatively rare resistant organisms [5]), have conspired over the past two decades to de-incentivize new antibiotic drug development. The scientific difficulties inherent in this therapeutic area are also unique [6]. For example, the chemical matter that dominates modern drug discovery in the pharmaceutical industry is generally tailored for activity on human tissues and cells [7]. Bacteria are completely different in terms of the physical chemical properties of compounds required to penetrate cells [8]; therefore, on-target activity and potency in biochemical assays often is not replicated in studies of whole cells. In retrospective analyses, both GlaxoSmithKline [9] and AstraZeneca [10] describe decades of sustained effort that resulted in no new drug leads. We are well within the resistance era, where new drugs are rare and resistance increases unabated [11]. The new reality that we must face is that the scientific, commercial, and regulatory conditions are not presently aligned for the discovery of new drug classes. In fact they may never be as perfectly positioned as they were during the Golden Age of antibiotic drug discovery in the decade that followed the Second World War [11]. As a result, while we must never abandon the search for new antibiotic drugs, we must also steadfastly focus on preserving our existing drugs. This can be accomplished in part with judicious management of current drugs [12]. For example, we can ensure that antibiotics are used exclusively for the treatment of bacterial infection, and work toward a ‘right drug for the right bug’ approach–for example, through innovation in rapid and reliable point-of-care diagnostics, currently unavailable to physicians who, more often, rely on empiric prescription [4,13]. Continuing and enhanced education of clinicians, farmers, and the public of the dangers of inappropriate antibiotic use is also vital. These stewardship strategies must be vigorously explored if we are to slow the progression toward a postantibiotic age.
Glossary Antibiotic adjuvant: nonantibiotic molecule that potentiates the activity of an antibiotic. Class I adjuvants: compounds that block antibiotic resistance. Class I.A adjuvants block active resistance, that is, specific inactivating enzymes, bypass mechanisms etc., Class I.B adjuvants overcome metabolic or physiologic barriers to antibiotic activity, e.g., facilitate membrane penetration, block biofilms, etc. Class II adjuvants: compounds that enhance antibiotic action by interacting with host defense mechanisms. Efflux pumps: proteins and protein complexes that actively (i.e., use energy) move antibiotics out of the cell against a concentration gradient.
Antibiotic Adjuvants A complimentary strategy to protect our existing drugs is through the use of antibiotic adjuvants (see Glossary), compounds that enhance the activity of current drugs and can minimize, and even directly block, resistance [14–16]. Adjuvants are delivered together with antibiotics and therefore are combination drugs (Figure 1). The concept of antibiotic adjuvants borrows from the success of antibiotic combinations (antibiotic A + antibiotic B) in the clinic [17,18]. These have been used for decades to achieve synergy, cover microbial spectrum, and suppress resistance [19]. Well known combinations include aminoglycoside + penicillin combinations (e.g., gentamicin + ampicillin) for treatment of enterococcal infections, trimethoprim + sulfamethoxazole that has efficacy versus a number of pathogens, and multidrug therapy for treatment of tuberculosis (Box 1). Such combinations have found particular use because synergistic interactions are achieved through a variety of mechanisms, including targeting of distinct steps in common or linked biochemical pathways, enhancement of uptake or suppression of efflux. The result is that efficacy is greater than the sum of the individual agents (synergy), generally resulting in faster kill times or enhanced killing, thereby limiting the opportunity for resistant organisms to emerge. The molecular basis of antibiotic synergy underscores the importance of understanding the mechanisms of antibiotic action, including primary and secondary targets in a variety of pathogens (not just model organisms), data that are generally poorly reported, making rational selection of antibiotic pairs difficult at present; however, systems biology approaches can be applied to this problem with success [20]. Nevertheless, in the absence of mechanistic data, machine-learning strategies have recently been used to predict the efficacy of combinations in the antifungal realm and to offer an opportunity to identify new antibacterial combinations as well [21].
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Examples
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O
Cl
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Loperamide
OH
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Figure 1. Antibiotic Adjuvants Can Be Divided into Two Classes. When given with an antibiotic (A), Class I adjuvants inhibit active or intrinsic (passive) antibiotic resistance in bacteria, while Class II adjuvants enhance the host (H) ability to kill bacteria. Class I adjuvants can be further subdivided into Class I.A that inhibit (I) active resistance mechanism, for example, inactivating enzymes, bypass mechanisms, efflux, that are generally imported by horizontal gene transfer, and Class I.B adjuvants are nonantibiotics (N) that block passive or intrinsic mechanisms including permeability barriers such as the outer membrane in Gram-negative bacteria, biofilms, etc.
In contrast to antibiotic combinations, antibiotic adjuvants [15,16,18,22] show little or no antimicrobial activity alone. Instead, when combined with drugs, they enhance antibiotic activity under specific conditions. It is useful to classify antibiotic adjuvants into two general classes based on target profile: Class I adjuvants that work with antibiotics on bacterial targets, and Class II adjuvants that enhance antibiotic activity in the host (Figure 1). Class I adjuvants can be further differentiated based on their mechanisms. Class I.A compounds directly inhibit antibiotic resistance (inactivating enzymes, efflux pump systems, or alternate targets) and are the only adjuvants in current clinical use. Class I.B adjuvants enhance antibiotic activity by circumventing intrinsic resistance mechanisms including metabolic pathways or physiology other than direct inhibition of specific resistance elements. By contrast, Class II adjuvants do not directly impact bacteria but rather operate on host properties to potentiate antibiotic action. Examples of Class I. B and Class II adjuvants have yet to be approved by regulatory agencies as formulated drug combinations with antibiotics, but are being explored in preclinical models.
Mechanisms of Antibiotic Resistance Resistance to antibiotics occurs through a variety of molecular mechanisms, including decreased drug permeability, active efflux, alteration or bypass of the drug target, production of antibiotic-modifying enzymes, and physiological states such as biofilms that are less susceptible to antibiotic activity (Figure 2). All of these mechanisms are susceptible to inhibition by small molecules and thus are potential targets for antibiotic adjuvants. For the purposes of targeting resistance with adjuvants, it is useful to classify resistance as either active, that is, mechanisms that have evolved specifically to detoxify specific antibiotics, or passive, mechanisms that are intrinsic to specific bacteria that have the effect of resistance but are not necessarily directly targeted to an individual antibiotic. Examples of active resistance include genes acquired by horizontal and vertical transfer and the upregulation of ‘silent’ chromosomal elements that can confer drug resistance to otherwise sensitive bacteria. These include most of
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Box 1. Antibiotic Combinations and Measurement of Synergy Combining antibiotics is a strategy often used by clinicians. The objective is to ensure coverage of potential bacterial pathogens and/or resistance profiles during empiric therapy, suppress the emergence of resistance, avoid toxicity (by using less drug), or to achieve synergy. The latter is especially useful in increasing rates of cell killing and managing difficult-to-treat infectious organisms. Multidrug therapy is common in the treatment of tuberculosis for example. The causative agent, Mycobacterium tuberculosis, is a slow-growing organism that is generally found as an intracellular pathogen. Achieving adequate antibiotic concentrations and maintaining these over a sufficient period of time to ensure cell death is very difficult with only one antibiotic. Instead, a combination of three or more drugs given simultaneously is standard practice. The molecular mechanisms of synergy in antibiotic combinations are similar to those in antibiotic adjuvant pairs. They include targeting different critical but nonredundant steps in metabolic pathways (e.g., trimethoprim + sulfamethoxazole that hit distinct steps in folate metabolism), enhanced uptake of drugs (e.g., penicillin + aminoglycoside combinations in enterococci), and inhibition of redundant enzymes (e.g., targeting of multiple penicillin-binding proteins by b-lactam antibiotics). Measuring synergy in both antibiotic pairs and with antibiotic adjuvants is easily accomplished using simple plate assays, where compounds are applied on separate paper disks and allowed to diffuse on agar plates inoculated with target bacteria. Synergy is indicated with enhanced killing at the interface of the diffusion zones. Quantitative measurement of synergy can be measured using the simple checkerboard strategy, where concentrations of both agents are systematically diluted to identify concentrations of both drugs that achieve the most potent interaction. A simple mathematical device, the fraction inhibitory concentration index (FICI) between two compounds, A and B, is calculated from the following formula: FICI
¼
MICðA in combination with BÞ MICðA aloneÞ
þ
MICðB in combination with AÞ MICðB aloneÞ
(1)
In principle, any value of FICI below 1 indicates interaction that is better than additive (i.e., synergistic); however, given the accepted one dilution error of MIC determinations, compounds are reported as synergistic when FIC <0.5, that is, a fourfold decrease in MIC. In the case of adjuvant molecules that do not show an MIC, it is common and useful to use the highest concentration tested and indicate that the FICI is less than the value obtained. Measuring FICI in cases where adjuvants have no measurable MIC poses a challenge for the quantification of adjuvant potency. By analogy to the 0.5 FICI value to define synergy, the concentration of the adjuvant that reduces MIC fourfold is a reasonable marker of potency. More relevant is the concentration of adjuvant that lowers the antibiotic MIC in resistant bacteria to equal to, or less than, the breakpoint concentration, that is, the concentration of antibiotic that defines whether a strain is resistant or sensitive. The Rescue Concentration (RC) is therefore defined as the concentration of adjuvant required to lower the effective concentration of the antibiotic to less than, or equal to, the breakpoint.
the commonly known resistance elements such as detoxifying enzymes (b-lactamases, aminoglycoside acetyltransferases and kinases, etc.), bypass and target alteration mechanisms (mec genes in Staphylococcus aureus, van genes in enterococci), and some efflux systems (tetA, qac, etc.). By contrast, passive resistance occurs through nonspecific mechanisms where the antibiotic target is nevertheless sensitive to the antibiotic. These include the broad-spectrum efflux systems found in most bacteria (e.g., acrAB/tolC in Escherichia coli), mutations in porins (e.g., carbapenem resistance through mutation of oprD in Pseudomonas aeruginosa), and changes in physiology such as growth in biofilms, where antibiotic susceptibility is greatly decreased. The differences between active and passive resistance are not always simple to define, for example, upregulation of intrinsic efflux systems, but the structure is useful when considering Class I adjuvants and their targets
Class I.A Antibiotic Adjuvants: Inhibitors of Active Resistance The most clinically successful adjuvants are the inhibitors of b-lactamases, enzymes that hydrolytically inactivate penicillin, cephalosporin, and carbapenem drugs by a ring-opening mechanism (Figure 3). b-Lactamases are widespread in bacterial pathogens, and are often acquired through horizontal gene transfer; they represent the most important antibiotic-resistance challenge today given the fact that these are the most widely used antibiotics in the clinic. b-Lactamases operate by one of two molecular mechanisms: enzymes that use an active site Ser residue to covalently capture the antibiotic then release the inactive compound after
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Modifying Enzymes
Efflux
Anbioc Physiology
Permeability
Target Bypass
Figure 2. Mechanisms of Resistance to Antibiotics. These include inactivating enzymes, efflux systems, decreased permeability, physiology such as biofilm formation, and target bypass or alteration.
hydrolysis (Figure 3, path i), or enzymes that use active-site Zn2+ atoms to activate a water molecule that is positioned for hydrolysis of the antibiotic. Ser-b-lactamases are further arranged in three types: A, C, and D based on protein fold and substrate specificity. Regardless of mechanism, the end result is the same, with hydrolytic opening of the b-lactam ring that is essential for antibiotic action. In the 1970s, clavulanic acid (Figure 3), a b-lactam-containing natural product, was isolated from a strain of Streptomyces clavuligerus that also produced a b-lactam antibiotic [23]. Clavulanic acid has poor antibiotic activity, but it does show potent and irreversible inactivation of Ser-b-lactamases [24]. This discovery led to the first antibiotic/adjuvant pair to be successfully approved for clinical use. Pairing of clavulanic acid with amoxicillin generated Augmentin, a combination drug that has been in clinical use for over 30 years. The success of clavulanic acid spurred efforts to develop other b-lactamase inhibitors, including the b-lactam sulfones tazobactam and sulbactam that are paired with penicillins, respectively piperacillin and ampicillin. More recently, a new class of b-lactamase inhibitor, the diazabicyclooctanes (DABCOs) was introduced into clinical practice. The DABCO avibactam, when paired with the cephalosporin ceftazidime, forms the drug Avycaz, approved for clinical use by the FDA in 2015. Clavulanic acid is predominantly restricted to type A Ser-b-lactamases, and after three decades of clinical use many resistant alleles are circulating in pathogens. The emergence of extended-spectrum b-lactamases (ESBLs) in pathogens over the past several years has further eroded the efficacy of clavulanic acid. The penicillanic acid sulfones, such as tazobactam, showed efficacy toward some of the common ESBLs in the 1990s, but have limited effectiveness with type A carbapenemases, the increasingly common type C b-lactamases such as plasmid-born AmpC, and the oxacillinases (type D). Avibactam on the other hand does show efficacy vs. type A ESBLs such as CTX-M, AmpCs, and type A carbapenemases such as Klebsiella pneumoniae carbapenemase (KPC) [25,26]. This strategy–to block Ser b-lactamases–continues to attract innovation and imitation. For example, combinations of avibactam with ceftaroline and aztreonam are in late-stage clinical trials, and the DABCO relebactam paired with imipenem/cilastatin is also being explored. Zerbaxa (ceftolozane/tazobactam), a combination
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General mechanism of Ser (i) and metallo- (ii) β-lactamases H
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Aspergillomarasmine A
Figure 3. b-Lactamases and Inhibitors. Ser-b-lactamases (path i) are the most common in the clinic and operate through the formation of a covalent complex with the b-lactam antibiotic. The only antibiotic adjuvants in clinical use are inhibitors of these enzymes. Metallo b-lactamases (path ii) cleave the b-lactam ring of the antibiotics using a Zn-activated water molecule. No antibiotic adjuvants are yet available for these enzymes, though aspergillomarasmine A is undergoing preclinical testing.
that is especially effective vs. Pseudomonas, was approved by the FDA in 2014. Carbavance, a combination of the first in class boronic acid vaborbactam (Figure 3) with meropenem, recently received fast-track status from the FDA. The Zn-dependent metallo (type B) b-lactamases, which efficiently confer resistance to all penicillins, cephalosporins, and carbapenems, are not inhibited by the b-lactamase inhibitors in current clinical use. Historically, metallo-b-lactamases have not been a pressing clinical problem as pathogens harboring such enzymes, while alarming, have been relatively rare. However, the incidence of pathogens expressing metallo-b-lactamases is increasing globally, and the emergence of NDM-1 in 2010, which is now broadly distributed in Enterobacteriaceae, Pseudomonas, and Acinetobacter strains, is a growing concern [27]. The fact that ndm alleles are invariantly associated with multiple resistance elements spanning most (sometimes all)
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available alternative antibiotics, including aminoglycosides and recently colistin, is generating concern for the emergence of pan-drug-resistant Gram-negative bacterial pathogens. Few inhibitors of metallo-b-lactamases that are effective in animal models of infection are known. Aspergillomarasmine A (Figure 3), a fungal natural product discovered in a cell-based screen for NDM-1 inhibitors, in combination with meropenem, does show efficacy in murine models of infection [28]. The compound rescues the antibiotic activity of meropenem in pathogens expressing NDM-1 and VIM metallo-b-lactamases and operates by sequestering a Zn2+ ion essential for catalytic activity and raises the possibility of clinical use as a new Class I.A antibiotic adjuvant. The strategy of directly targeting antibiotic-resistance mechanisms with adjuvants is applicable to other resistance mechanisms and antibiotics, but thus far examples of clinical use are limited to the b-lactamases. Aminoglycoside antibiotics are bactericidal drugs that target the ribosome; they are highly effective against both Gram-negative and Gram-positive pathogens. Compounds such as gentamicin, tobramycin, and amikacin continue to find clinical use despite widespread and diverse resistance. Prevalent mechanisms include inactivating enzymes (kinases, acetyltransferases, and adenylyltransferases) and ribosome methylation [29]. Inhibitors of aminoglycoside kinases (APHs) have been reported that inhibit the enzymes and rescue antibiotic activity in resistant cells. The similarity of APHs to eukaryotic protein kinases in both structure and mechanism (despite an absence of protein sequence identity) inspired a survey of protein kinase inhibitors as potential inhibitors of APHs [30]. Several candidates were identified, but one scaffold in particular, the pyrazolopyrimidines that are human Src and PI3 kinases, inhibit and bind to APH(30 )-I (a dominant resistance mechanism in Gram-negative pathogens) in a fashion that is distinct from the binding mode in human kinases, offering a potential route for derivatives that selectively inhibit APH over human targets [31]. Other examples of blocking aminoglycoside resistance with antibiotic adjuvants include tropolones that inhibit aminoglycoside adenlylytransferases [32,33] and cationic peptides that target both APHs and acetyltansferases [34].
Class I.B Antibiotic Adjuvants: Inhibitors of Passive Resistance Bacteria can be insensitive to antibiotics because of intrinsic genetic network organization, physiology, or through the presence of nonspecific evasion mechanisms such as broad specificity efflux systems. All of these are potential targets for adjuvants. While the strategy to identify adjuvants for active resistance is relatively straightforward since the resistance targets are commonly known–for example, a b-lactamase that can be screened for inhibition using purified enzyme–adjuvants for countering passive resistance generally require cell-based screens for antibiotic potentiation followed by elucidation of mechanism (e.g., a screen for adjuvants of the semi-synthetic tetracycline antibiotic minocycline in P. aeruginosa identified several candidates, including loperamide (Imodium) [35]). Additional experiments revealed that the mode of action of loperamide, which has no antibiotic activity, was to decrease the electrical component of the proton motive force in Gram-negative bacteria. To counter this effect and maintain ATP synthesis levels, the bacteria increase the pH gradient across the inner membrane. The increase in DpH, in turn, increases uptake of tetracycline antibiotics, thereby overcoming intrinsic resistance. This observation inspired a screen for additional compounds that perturb the proton motive force in bacteria, identifying several adjuvant molecules [36]. This approach of phenotypic whole-cell screening for enhancement of antibiotic activity has revealed several adjuvants, including inhibitors of wall teichoic acid biosynthesis in methicillinresistant S. aureus (MRSA) [37–39], compounds that perturb cell shape [40], murgocil (an inhibitor of the peptidoglycan biosynthesis enzyme MurG) [20], and inhibitors of efflux systems [41]. Melander has demonstrated, through whole-cell phenotypic assays, that that 2-aminoimidazole derivatives can suppress b-lactamase activity in K. pneumoniae [42], MRSA [43], and multidrug-resistant Gram negative pathogens [44] through as yet unknown mechanisms.
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Inhibition of efflux offers a powerful strategy to enhance the activity of antibiotics. The most common antibiotic-efflux systems in bacterial pathogens are the major facilitator superfamily (MFS) and the tripartite RND systems found in many Gram-negative bacteria. These generally have broad specificities that enable active efflux of many antibiotic classes [45]. PAbN was one of the first inhibitors of RND efflux systems active in a variety of bacteria, including P. aeruginosa, which has a number of redundant efflux systems [46]. As a potent inhibitor of efflux, PAbN enhances the antibiotic activity of a number of antibiotics from different structural classes, demonstrating that such inhibitors can act as broad-spectrum adjuvants. Furthermore, combining PAbN with the fluoroquinolone antibiotic levofloxacin decreased the emergence of resistance by more than four orders of magnitude, demonstrating the value of adjuvants in suppressing the emergence of resistance [46]. Challenges with toxicity of PAbN and other efflux inhibitors have thus far prevented their use in the clinic; however, the approach is sound and it deserves increased attention. Most bacteria can readily form biofilms, and in this physiological form they are highly tolerant to antibiotics. Since biofilms are a significant problem in the clinic (e.g., indwelling medical devices, epithelial linings, etc.) compounds that enhance antibiotic activity toward biofilms have great promise as adjuvant compounds. Adjuvant molecules have the potential to act at several points in biofilm development, including prevention of biofilm formation [47–49], enhancement of antibiotic activity in mature biofilms [50], and dispersal of biofilms releasing planktonic cells that are sensitive to drugs [49,51,52].These examples reveal the depth and breadth of adjuvant targets in bacterial cells uncovered in small-molecule screens. This is supported by genetic studies of chemical synthetic lethality in E. coli. Miller and coworkers have systematically interrogated the Keio collection of E. coli BW25113 and uncovered dozens of non-obvious gene deletions that enhanced the activity of antibiotics [53]. These nonessential genes are obvious targets for antibiotic adjuvants. In particular, 61 gene deletion strains showed increased sensitivity to eight or more antibiotics. Recently, this approach has been significantly expanded and improved by Brown and colleagues using a high-throughput quantitative growth kinetics platform that identifies both subtle and strong antibiotic–genetic interactions that expand chemical genetic space by 50– 200% depending on the antibiotic interrogated [54]. This work is identifying a very rich adjuvant target landscape, and campaigns that systematically explore the drugability of these targets could prove highly effective in identifying Class I.B antibiotic adjuvants.
Class II Adjuvants: Targeting Host Defense Mechanisms Enhancing host defense mechanisms offers an alternative set of targets for antibiotic adjuvants. Immunomodulatory peptides such as LL-37 have long been known to enhance antimicrobial activity of the innate immune system, and several peptides–both with and without direct antimicrobial activity–have been in, or are in, clinical trials (reviewed in [55]). These can synergize with antibiotics as Class I adjuvants but also have the potential to act through host defense systems as Class II adjuvants [56]. Given the tremendous depth of knowledge and effort in the immunomodulatory peptide field, these likely offer the greatest potential for adjuvant-antibiotic therapy target at the host in the short term. Strategies to explore small molecule enhancement of antibiotic action in the host are worthy of exploration. For example, a screen of microbial natural product extracts for enhancers of macrophage killing activity vs. Streptococcus mutans identified the neutral compound streptazolin [57]. Mechanistic analysis revealed that streptazolin stimulates macrophage activity through the phosphoinositide 3-kinase pathway resulting in upregulation of nuclear factorkB (NF-kB). Such a screening strategy is promising as a means to identify additional Class II antibiotic adjuvants. The caveat here is the need for balance between immune modulation to increase antibiotic activity vs. the pathogen, while sparing the host the deleterious effects of overactivation of the immune system.
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Concluding Remarks
Outstanding Questions
Antibiotic resistance is one of the most pressing issues facing medicine today. New drugs candidates are increasingly difficult to identify and bring to market. As a result there is a growing gap between clinical need and drug innovation. Furthermore, the history of antibiotic discovery and use predicts that, even if we had access to a rich pipeline of drug candidates similar to those used in the past, that is, broad-spectrum single agents, resistance will continue to be a significant challenge. Antibiotic adjuvants offer an orthogonal and complementary strategy to new antibiotic discovery (see Outstanding Questions). These compounds can enhance and preserve the activity of our existing drug arsenal. The efficacy of b-lactamase inhibitors in the clinic over the past decades is proof that the concept is robust and valuable. Furthermore, by directly targeting resistance or otherwise enhancing antibiotic activity, the impact and emergence of resistance can be minimized (though never eliminated). In this new era of antibiotic resistance, we must continue to explore new strategies to preserve our antibiotic drugs and the adjuvant concept is one contribution that has great promise.
Can non-b-lactamase Class I.A adjuvants find use in the clinic? What molecular epidemiological studies are necessary to provide a suitable value proposition?
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