The Science of Antibiotic Discovery

We are experiencing an antimicrobial resistance (AMR) crisis, brought on by the drying up of the antibiotic discovery pipeline and the resulting unchecked spread of resistant pathogens. Traditional methods of screening environmental isolates or compound libraries have not produced a new drug in over 30 years. Antibiotic discovery is uniquely difficult due to a highly restrictive penetration barrier and other mechanisms that allow bacteria to survive in the presence of toxic compounds. In this Perspective, we analyze the challenges facing discovery and discuss an emerging new platform for antibiotic discovery. The penetration barrier makes screening conventional synthetic compound libraries largely impractical, and actinomycetes, the main source of natural product compounds, have been overmined. The emerging platform is based on understanding the rules that guide the permeation of molecules into bacteria and on advances in microbiology, which enable us to identify and access attractive groups of secondary metabolite producers. Establishing this platform will enable reliable production of lead compounds to combat AMR. We are experiencing an antimicrobial resistance (AMR) crisis, brought on by the drying up of the antibiotic discovery pipeline and the resulting unchecked spread of resistant pathogens. Traditional methods of screening environmental isolates or compound libraries have not produced a new drug in over 30 years. Antibiotic discovery is uniquely difficult due to a highly restrictive penetration barrier and other mechanisms that allow bacteria to survive in the presence of toxic compounds. In this Perspective, we analyze the challenges facing discovery and discuss an emerging new platform for antibiotic discovery. The penetration barrier makes screening conventional synthetic compound libraries largely impractical, and actinomycetes, the main source of natural product compounds, have been overmined. The emerging platform is based on understanding the rules that guide the permeation of molecules into bacteria and on advances in microbiology, which enable us to identify and access attractive groups of secondary metabolite producers. Establishing this platform will enable reliable production of lead compounds to combat AMR. Discovering new antibiotics is uniquely challenging (Brown and Wright, 2016Brown E.D. Wright G.D. Antibacterial drug discovery in the resistance era.Nature. 2016; 529: 336-343Crossref PubMed Scopus (916) Google Scholar, Lewis, 2013Lewis K. Platforms for antibiotic discovery.Nat. Rev. Drug Discov. 2013; 12: 371-387Crossref PubMed Scopus (795) Google Scholar). In all other therapeutic areas, there is a positive correlation between accumulated knowledge and the ability to discover new drugs. This seemingly obvious relationship is paradoxically inverted for antibiotics. The field once enjoyed a golden era of discovery, ushered in by the Waksman platform (Lewis, 2012Lewis K. Antibiotics: Recover the lost art of drug discovery.Nature. 2012; 485: 439-440Crossref PubMed Scopus (152) Google Scholar). In the 1940s, Selman Waksman screened soil actinomycetes for their ability to produce zones of growth inhibition on a Petri dish overlaid with a test pathogen. This led to the discovery of streptomycin (Schatz et al., 1944Schatz A. Bugie E. Waksman S.A. Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria.Proc. Soc. Exp. Biol. Med. 1944; 55: 66-69Crossref Scopus (479) Google Scholar), and shortly afterward, to large-scale screening campaigns by industry. The golden era was launched, and the main classes of antibiotics were discovered in a fairly short period of time (Figure 1). The golden era ended rather abruptly in the early 1960s just as knowledge on mechanisms of antibiotic action and the nature of resistance began to accumulate. The once reliable Waksman platform was now increasingly rediscovering known compounds—aminoglycosides, tetracyclines, β-lactams, chloramphenicols, and macrolides (Figure 1). The low-hanging fruit—antibiotics commonly present in various species of actinomycetes—was overmined. At about the same time, there was considerable success in discovering synthetic antimicrobials. A small arsenal of synthetic compounds (isoniazid, pyrazinamide, and ethambutol) was identified by whole cell screening to fight the most important disease of the time, tuberculosis. A broad-spectrum compound, metronidazole, with excellent bactericidal activity was discovered, though limited to pathogens that live under anaerobic or microaerophilic conditions. Finally, there was an important, if accidental breakthrough in 1960s, when nalidixic acid, a side-product of a synthetic pathway leading to chloroquine, was found to kill Escherichia coli. Nalidixic acid was not particularly potent, but its fluorinated analogs became the fluoroquinolones that target DNA gyrase and topoisomerase, and are one of the most successful classes of broad-spectrum antibiotics, second only to β-lactams. Apart from the discovery of fluoroquinolones, synthetic chemistry was also highly successful in introducing increasingly effective analogs that converted narrow-spectrum compounds acting against Gram-positive species into broad-spectrum antibiotics (penicillin-ampicillin; erythromycin-azithromycin) and analogs active against resistant pathogens. Things were looking up for synthetic compounds and increasingly dim for natural products. It seemed that a proper focus on synthetics would solve the emerging problem of resistance. The industry combined several approaches to produce a high-tech discovery platform: genomics and proteomics to identify numerous essential targets; combinatorial chemistry to produce millions of compounds; and robotic high-throughput screening against whole cells or isolated targets to discover lead antimicrobials. Rational optimization of leads informed by the crystal structure with a target would then produce new classes of antibiotics. This approach has proven to be quite successful in various therapeutic areas. This well thought-through platform, however, failed for antimicrobials. Very large screening campaigns against dozens of targets did not produce reasonable leads. To their credit, scientists from GlaxoSmithKline and then Astra Zeneca published detailed accounts of this negative experience (Payne et al., 2007Payne D.J. Gwynn M.N. Holmes D.J. Pompliano D.L. Drugs for bad bugs: confronting the challenges of antibacterial discovery.Nat. Rev. Drug Discov. 2007; 6: 29-40Crossref PubMed Scopus (1798) Google Scholar, Tommasi et al., 2015Tommasi R. Brown D.G. Walkup G.K. Manchester J.I. Miller A.A. ESKAPEing the labyrinth of antibacterial discovery.Nat. Rev. Drug Discov. 2015; 14: 529-542Crossref PubMed Scopus (300) Google Scholar). Synthetic compounds were running into a barrier. The barrier is the bacterial cell envelope evolved to restrict toxic compounds from entering the cell. This barrier is especially effective in Gram-negative bacteria that have an additional, outer membrane. The surface of the outer membrane is made of lipopolysaccharide (LPS), which is negatively charged and forms a hydrophilic network stabilized by divalent cations. LPS prevents the penetration of large and hydrophobic compounds. The inner, or cytoplasmic membrane, has the conventional composition and is made of a phospholipid bilayer and membrane proteins. The hydrophobic lipid bilayer restricts the penetration of hydrophilic compounds, and the result is an almost impenetrable barrier. Compounds that leak across the barrier are extruded by transenvelope multidrug resistance (MDR) pumps that recognize their substrates by their hydrophobic properties, which distinguish them from the hydrophilic cellular metabolites (Li et al., 2015Li X.Z. Plésiat P. Nikaido H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria.Clin. Microbiol. Rev. 2015; 28: 337-418Crossref PubMed Scopus (602) Google Scholar, Lomovskaya and Lewis, 1992Lomovskaya O. Lewis K. Emr, an Escherichia coli locus for multidrug resistance.Proc. Natl. Acad. Sci. USA. 1992; 89: 8938-8942Crossref PubMed Scopus (294) Google Scholar). Nutrients cross through outer membrane porins and specialized transporters (Figure 2). In this Perspective, we consider knowledge-based approaches to revive antibiotic discovery. We will discuss the stratification of antimicrobial classes among microorganisms and identifying attractive producers outside of the overmined actinomycetes, uncultured bacteria and silent operons, the need for anti-persister compounds to treat chronic infections, the opportunity to design membrane-acting agents, rules of permeation that govern penetration of compounds into bacteria, rational approaches to combat drug resistance, an emerging platform combining natural product discovery and medicinal chemistry, and the troubled relationship between discovery and development of antibiotics. The field of anti-infectives has been working without a discovery platform for the past 50 years, during which time pathogens continued to accumulate resistance to existing antibiotics. The result of this disbalance, not surprisingly, is the antimicrobial resistance crisis (AMR). The World Health Organization (WHO) recently introduced a list of priority pathogens (Tacconelli et al., 2018Tacconelli E. Carrara E. Savoldi A. Harbarth S. Mendelson M. Monnet D.L. Pulcini C. Kahlmeter G. Kluytmans J. Carmeli Y. et al.WHO Pathogens Priority List Working GroupDiscovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis.Lancet Infect. Dis. 2018; 18: 318-327Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar) and noted those of “critical priority”—drug-resistant Enterobacteriaceae (E. coli, Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter), Pseudomonas aeruginosa, and Acinetobacter baumannii, which are responsible for a global health problem. All of the critical priority pathogens are Gram-negative bacteria. We now have isolates of A. baumannii, for example, that are resistant to all available antibiotics (Göttig et al., 2014Göttig S. Gruber T.M. Higgins P.G. Wachsmuth M. Seifert H. Kempf V.A.J. Detection of pan drug-resistant Acinetobacter baumannii in Germany.J. Antimicrob. Chemother. 2014; 69: 2578-2579Crossref PubMed Scopus (63) Google Scholar). K. pneumoniae is of particular concern—infection with carbapenem-resistant strains has a mortality rate of 30%–40% in the United States and 40%–50% in Europe (Ramos-Castañeda et al., 2018Ramos-Castañeda J.A. Ruano-Ravina A. Barbosa-Lorenzo R. Paillier-Gonzalez J.E. Saldaña-Campos J.C. Salinas D.F. Lemos-Luengas E.V. Mortality due to KPC carbapenemase-producing Klebsiella pneumoniae infections: Systematic review and meta-analysis: Mortality due to KPC Klebsiella pneumoniae infections.J. Infect. 2018; 76: 438-448Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, Xu et al., 2017Xu L. Sun X. Ma X. Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae.Ann. Clin. Microbiol. Antimicrob. 2017; 16: 18Crossref PubMed Scopus (147) Google Scholar). The AMR crisis threatens the return of the pre-antibiotic era of epidemics and pandemics. It is important to note that antibiotics not only save lives, but enable modern medicine. Without antibiotics, surgery, chemotherapy, or organ transplantation become highly problematic. Antibiotic resistance comes in many forms, and all logical possibilities seem to have been realized in nature—restricted penetration, efflux, target modification, destruction/modification of the antibiotic, target switching, and target sequestration (Alekshun and Levy, 2007Alekshun M.N. Levy S.B. Molecular mechanisms of antibacterial multidrug resistance.Cell. 2007; 128: 1037-1050Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar, Lewis, 2013Lewis K. Platforms for antibiotic discovery.Nat. Rev. Drug Discov. 2013; 12: 371-387Crossref PubMed Scopus (795) Google Scholar). Many resistance determinants, most notably β-lactamases that provide resistance to β-lactam antibiotics, travel on plasmids and can rapidly spread. Apart from resistance, there is also antibiotic tolerance that is primarily responsible for recalcitrance of chronic infections that we discuss in a section of this Perspective. The main approach to AMR has been a push to introduce more antibiotics. While this is obvious, we also discuss strategies specifically aimed at limiting or eliminating resistance (see section “Combatting Resistance”). Let us consider the current pipeline for novel antimicrobials; this is the best measure for the health of the discovery enterprise. The most successful direction has been, not surprisingly, discovery and development of novel inhibitors of β-lactamases, enzymes that destroy β-lactam antibiotics, the penicillins and cephalosporins. Clavulanic acid and tazobactam that inhibit many serine β-lactamases (SBLs) are used in combination with amoxicillin and piperacillin, respectively. These combinations have been a mainstay of therapy for decades. However, they have been losing their effectiveness after pathogens acquired a number of SBLs that are not susceptible to inhibition by clavulanic acid and tazobactam, in particular the K. pneumoniae carbapenemase (KPC). Several new synthetic inhibitors active against KPC were recently introduced in clinical practice. They include avibactam (Levasseur et al., 2014Levasseur P. Girard A.M. Lavallade L. Miossec C. Pace J. Coleman K. Efficacy of a Ceftazidime-Avibactam combination in a murine model of Septicemia caused by Enterobacteriaceae species producing ampc or extended-spectrum β-lactamases.Antimicrob. Agents Chemother. 2014; 58: 6490-6495Crossref PubMed Scopus (24) Google Scholar) and relebactam (Blizzard et al., 2014Blizzard T.A. Chen H. Kim S. Wu J. Bodner R. Gude C. Imbriglio J. Young K. Park Y.W. Ogawa A. et al.Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®.Bioorg. Med. Chem. Lett. 2014; 24: 780-785Crossref PubMed Scopus (102) Google Scholar), as well as vaborbactam, a boron-containing inhibitor (Hecker et al., 2015Hecker S.J. Reddy K.R. Totrov M. Hirst G.C. Lomovskaya O. Griffith D.C. King P. Tsivkovski R. Sun D. Sabet M. et al.Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases.J. Med. Chem. 2015; 58: 3682-3692Crossref PubMed Scopus (218) Google Scholar). These compounds, however, are not active against the newly emerged metallo β-lactamases, such as NDM-1, or against the SBL carbapenemase OXA-23 from A. baumannii. A natural product, aspergillomarasmin, has been recently shown to be active against NDM-1 (King et al., 2014King A.M. Reid-Yu S.A. Wang W. King D.T. De Pascale G. Strynadka N.C. Walsh T.R. Coombes B.K. Wright G.D. Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance.Nature. 2014; 510: 503-506Crossref PubMed Scopus (299) Google Scholar). Taniborbactam (Liu et al., 2019Liu B. Trout R.E.L. Chu G.H. McGarry D. Jackson R.W. Hamrick J.C. Daigle D.M. Cusick S.M. Pozzi C. De Luca F. et al.Discovery of Taniborbactam (VNRX-5133): A Broad-Spectrum Serine- and Metallo-beta-lactamase Inhibitor for Carbapenem-Resistant Bacterial Infections.J. Med. Chem. 2019; (Published online December 16, 2019)https://doi.org/10.1021/acs.jmedchem.9b01518Crossref Scopus (64) Google Scholar) and durlobactam (Durand-Réville et al., 2017Durand-Réville T.F. Guler S. Comita-Prevoir J. Chen B. Bifulco N. Huynh H. Lahiri S. Shapiro A.B. McLeod S.M. Carter N.M. et al.ETX2514 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii.Nat. Microbiol. 2017; 2: 17104Crossref PubMed Scopus (111) Google Scholar) are active against NDM-1 and OXA-23, respectively, and are in clinical development. There will be more challenges ahead as well, because β-lactamases come in a multitude of forms (Bush and Jacoby, 2010Bush K. Jacoby G.A. Updated functional classification of beta-lactamases.Antimicrob. Agents Chemother. 2010; 54: 969-976Crossref PubMed Scopus (1272) Google Scholar), and there is little doubt that Nature will provide additional ones to the pathogens. Even effective β-lactamase inhibitors, however, are not foolproof. Some of the resistance to β-lactams is target-based. These compounds inhibit PBPs that are transpeptidases that help build the peptidoglycan cell wall (Figure 2). Mutations in the PBPs, or variants of these proteins borrowed by plasmid acquisition, can provide resistance to β-lactams. In the most challenging category—novel compound/novel target (or novel mode of action against a known target)—the pipeline is thin. Let us consider leads that are either in clinical trials or have a realistic potential to enter into development—compounds with efficacy in a mouse thigh model. In this model, animals are made neutropenic (production of white blood cells is disrupted by treatment with cyclophosphamide), and antibiotics are assessed by their ability to kill or prevent propagation of the pathogen in the absence of an active immune response. This is a challenging model and has proved to be a reasonable predictor for the developmental potential of a compound. The need for novel compounds acting against Gram-positive bacteria is considered to be relatively low, and the pharmaceutical industry has limited interest in developing them; the few novel compounds in this category are being developed primarily by biotechnology companies (Table 1).Table 1Novel Compounds Hitting Novel Targets in DevelopmentCompoundTarget/StageTarget PathogenDeveloperReferenceGepotidacintype II topoisomerase (new site on A subunit)/phase 3S. aureusGlaxoSmithKlineScangarella-Oman et al., 2020Scangarella-Oman N.E. Ingraham K.A. Tiffany C.A. Tomsho L. Van Horn S.F. Mayhew D.N. Perry C.R. Ashton T.C. Dumont E.F. Huang J. et al.In vitro activity and microbiological efficacy of gepotidacin from a phase 2, randomized, multicenter, dose-ranging study in patients with acute bacterial skin and skin structure infections.Antimicrob. Agents Chemother. 2020; 64 (e01302-19)PubMed Google ScholarZoliflodacintype II topoisomerase (B subunit)/phase 3N. gonorrhoeaeEntasis TherapeuticsTaylor et al., 2018Taylor S.N. Marrazzo J. Batteiger B.E. Hook 3rd, E.W. Seña A.C. Long J. Wierzbicki M.R. Kwak H. Johnson S.M. Lawrence K. Mueller J. Single-Dose Zoliflodacin (ETX0914) for Treatment of Urogenital Gonorrhea.N. Engl. J. Med. 2018; 379: 1835-1845Crossref PubMed Scopus (77) Google ScholarMurepavadinLptD/phase 3 (discontinued)aPhase III discontinued due to kidney failure in some patients; development of an inhaled formulation continues.P. aeruginosaPolyphorSrinivas et al., 2010Srinivas N. Jetter P. Ueberbacher B.J. Werneburg M. Zerbe K. Steinmann J. Van der Meijden B. Bernardini F. Lederer A. Dias R.L. et al.Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.Science. 2010; 327: 1010-1013Crossref PubMed Scopus (361) Google ScholarAfabicin (Debio 1450; AFN 1252)FabI/phase 2S. aureusDebiopharmKaplan et al., 2012Kaplan N. Albert M. Awrey D. Bardouniotis E. Berman J. Clarke T. Dorsey M. Hafkin B. Ramnauth J. Romanov V. et al.Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor.Antimicrob. Agents Chemother. 2012; 56: 5865-5874Crossref PubMed Scopus (76) Google ScholarBrilacidinmembrane-acting defensin mimetic/phase 2bNot clear if it is still in development.S. aureusInnovation PharmaceuticalsScott and Tew, 2017Scott R.W. Tew G.N. Mimics of Host Defense Proteins; Strategies for Translation to Therapeutic Applications.Curr. Top. Med. Chem. 2017; 17: 576-589Crossref PubMed Scopus (39) Google ScholarTeixobactinLipid II, III/preclinicalG+NovoBioticLing et al., 2015Ling L.L. Schneider T. Peoples A.J. Spoering A.L. Engels I. Conlon B.P. Mueller A. Schäberle T.F. Hughes D.E. Epstein S. et al.A new antibiotic kills pathogens without detectable resistance.Nature. 2015; 517: 455-459Crossref PubMed Scopus (1317) Google ScholarG0775 (arylomycin analog)LepB/preclinicalG−GenentechSmith et al., 2018Smith P.A. Koehler M.F.T. Girgis H.S. Yan D. Chen Y. Chen Y. Crawford J.J. Durk M.R. Higuchi R.I. Kang J. et al.Optimized arylomycins are a new class of Gram-negative antibiotics.Nature. 2018; 561: 189-194Crossref PubMed Scopus (124) Google ScholarPolymyxin/murepavadin-type chimera, POL7306BamA and LPS/preclinicalcProgression towards clinical studies interrupted due to insufficient therapeutic margins. Compounds are limited to those published in peer-reviewed journals.G−PolyphorLuther et al., 2019Luther A. Urfer M. Zahn M. Müller M. Wang S.Y. Mondal M. Vitale A. Hartmann J.B. Sharpe T. Monte F.L. et al.Chimeric peptidomimetic antibiotics against Gram-negative bacteria.Nature. 2019; 576: 452-458Crossref PubMed Scopus (80) Google ScholarDarobactinBamA/preclinicalG−Northeastern UniversityImai et al., 2019Imai Y. Meyer K.J. Iinishi A. Favre-Godal Q. Green R. Manuse S. Caboni M. Mori M. Niles S. Ghiglieri M. et al.A new antibiotic selectively kills Gram-negative pathogens.Nature. 2019; 576: 459-464Crossref PubMed Scopus (121) Google ScholarOdilorhabdinribosome/preclinicalG−Nosopharm SASPantel et al., 2018Pantel L. Florin T. Dobosz-Bartoszek M. Racine E. Sarciaux M. Serri M. Houard J. Campagne J.M. de Figueiredo R.M. Midrier C. et al.Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.Mol. Cell. 2018; 70: 83-94Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Zhao et al., 2018Zhao M. Lepak A.J. Marchillo K. VanHecker J. Andes D.R. In Vivo Pharmacodynamic Characterization of a Novel Odilorhabdin Antibiotic, NOSO-502, against Escherichia coli and Klebsiella pneumoniae in a Murine Thigh Infection Model.Antimicrob. Agents Chemother. 2018; 62: e01067-18Crossref PubMed Google ScholarTridecaptin M152-P3lipid IIG−CSIR-Institute of Microbial TechnologyJangra et al., 2019Jangra M. Kaur M. Tambat R. Rana R. Maurya S.K. Khatri N. Ghafur A. Nandanwar H. Tridecaptin M, a New Variant Discovered in Mud Bacterium, Shows Activity against Colistin- and Extremely Drug-Resistant Enterobacteriaceae.Antimicrob. Agents Chemother. 2019; 63: e00338-19Crossref PubMed Scopus (13) Google ScholarRetinoidmembraneS. aureusBrown UniversityKim et al., 2018Kim W. Zhu W. Hendricks G.L. Van Tyne D. Steele A.D. Keohane C.E. Fricke N. Conery A.L. Shen S. Pan W. et al.A new class of synthetic retinoid antibiotics effective against bacterial persisters.Nature. 2018; 556: 103-107Crossref PubMed Scopus (156) Google Scholara Phase III discontinued due to kidney failure in some patients; development of an inhaled formulation continues.b Not clear if it is still in development.c Progression towards clinical studies interrupted due to insufficient therapeutic margins. Compounds are limited to those published in peer-reviewed journals. Open table in a new tab Debiopharm is developing afabicin (in phase 2), a synthetic inhibitor of FabI, an essential enzyme in the fatty acid biosynthesis pathway, to target Staphylococcus aureus, which are Gram-positive (Kaplan et al., 2012Kaplan N. Albert M. Awrey D. Bardouniotis E. Berman J. Clarke T. Dorsey M. Hafkin B. Ramnauth J. Romanov V. et al.Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor.Antimicrob. Agents Chemother. 2012; 56: 5865-5874Crossref PubMed Scopus (76) Google Scholar). Resistance to this single-target antibiotic is a potential issue. Of particular interest is teixobactin, the first lead compound discovered in a screen of uncultured bacteria (Ling et al., 2015Ling L.L. Schneider T. Peoples A.J. Spoering A.L. Engels I. Conlon B.P. Mueller A. Schäberle T.F. Hughes D.E. Epstein S. et al.A new antibiotic kills pathogens without detectable resistance.Nature. 2015; 517: 455-459Crossref PubMed Scopus (1317) Google Scholar). Teixobactin is produced by Eleftheria terrae and has excellent activity against all tested Gram-positive bacteria, with a minimal inhibitory concentration (MIC) of 0.2 μg/mL against methicillin-resistant S. aureus (MRSA) and 0.06 μg/mL against Bacillus anthracis. It is highly efficacious in septicemia, lung, and mouse thigh infection models with several pathogens. The most remarkable, and unexpected property of teixobactin is the lack of any detectable resistance to this compound. Teixobactin hits two related targets—lipid II, precursor of peptidoglycan (Figure 2), and lipid III, precursor of wall teichoic acid (Homma et al., 2016Homma T. Nuxoll A. Gandt A.B. Ebner P. Engels I. Schneider T. Götz F. Lewis K. Conlon B.P. Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis.Antimicrob. Agents Chemother. 2016; 60: 6510-6517Crossref PubMed Scopus (54) Google Scholar). The targets are not mutable—they are not proteins and are not directly coded by DNA (see also the section on combatting resistance). In spite of a very substantial effort from industry and academia, there are only a few novel compounds in development that are active against Gram-negative bacteria (Figure 3; Table 1). The first to be discovered in this short list is murepavadin, a peptidomimetic (Melchers et al., 2019Melchers M.J. Teague J. Warn P. Hansen J. Bernardini F. Wach A. Obrecht D. Dale G.E. Mouton J.W. Pharmacokinetics and Pharmacodynamics of Murepavadin in Neutropenic Mouse Models.Antimicrob. Agents Chemother. 2019; 63: e01699-18Crossref PubMed Scopus (8) Google Scholar, Srinivas et al., 2010Srinivas N. Jetter P. Ueberbacher B.J. Werneburg M. Zerbe K. Steinmann J. Van der Meijden B. Bernardini F. Lederer A. Dias R.L. et al.Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.Science. 2010; 327: 1010-1013Crossref PubMed Scopus (361) Google Scholar). The goal of the project was to find improved versions of protegrin, a membrane-acting antimicrobial peptide from mammalian leukocytes. A screen against P. aeruginosa identified a highly potent compound, but unexpectedly, selection for resistant mutants showed that its target is not the membrane, but LptD, an essential protein of the outer membrane and a translocator of LPS (Figure 2). This accidental discovery produced the first lead against an outer membrane protein target. The initial hit was optimized into murepavadin with exceptional activity against P. aeruginosa, MIC 0.06 μg/mL. The compound, however, lacked activity against other bacteria. Nevertheless, given the importance of this pathogen, polyphor advanced murepavadin into clinical trials. Unfortunately, a phase 3 trial testing murepavadin for P. aeruginosa lung infection had to be terminated due to kidney damage in over half of the patients. Murepavadin is a polycation with 7 positive charges. Nephrotoxicity is a known liability of polycations such as polymyxin, a last-resort antibiotic that has considerable side-effects, due in part to accumulation in the acidic lysosomes of kidney cells (Nagai and Takano, 2004Nagai J. Takano M. Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity.Drug Metab. Pharmacokinet. 2004; 19: 159-170Crossref PubMed Scopus (206) Google Scholar). Murepavadin is unusually potent, and it is possible that a different regiment of administration will result in a better therapeutic window that will lead to its development into a drug. There are two essential proteins in the outer membrane—LptD and BamA. These are of prime interest as targets, because compounds binding to them do not have to cross the cell envelope barrier. The BamA protein, part of the BAM complex (Figure 2), is a chaperone and translocator that folds and inserts β-barrel proteins, such as porins, into the outer membrane (Konovalova et al., 2017Konovalova A. Kahne D.E. Silhavy T.J. Outer Membrane Biogenesis.Annu. Rev. Microbiol. 2017; 71: 539-556Crossref PubMed Scopus (104) Google Scholar). BamA, itself, is a β-barrel protein and lacks a well-structured catalytic center that typically serves as a target for drugs. Previous attempts to find small molecules that target BamA have not been successful, and a group from Genentech took a different approach, developing an antibody that binds BamA and inhibits growth of E. coli with defective LPS (Storek et al., 2018Storek K.M. Auerbach M.R. Shi H. Garcia N.K. Sun D. Nickerson N.N. Vij R. Lin Z. Chiang N. Schneider K. et al.Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal.Proc. Natl. Acad. Sci. USA. 2018; 115: 3692-3697Crossref PubMed Scopus (63) Google Scholar). In a recent study, a small molecule synthetic compound MRL-494 was reported to act against BamA, importantly, without the need to penetrate across the outer membrane (Hart et al., 2019Hart E.M. Mitchell A.M. Konovalova A. Grabowicz M. Sheng J. Han X. Rodriguez-Rivera F.P. Schwaid A.G. Malinverni J.C. Balibar C.J. et al.A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.Proc. Natl. Acad. Sci. USA. 2019; 116: 21748-21757Crossref PubMed Scopus (34) Google Scholar). MRL-494 is active against E. coli and K. pneumoniae, but there is also off-target activity as it acts against Gram-positive bacteria by disrupting their cytoplasmic membrane. An interesting drug lead against BamA was recently discovered by accident. In a search for compounds with a broader spectrum, the murepavadin scaffold was linked to polymyxin. Unexpectedly, this chimeric molecule bound not LptA, but LPS and BamA. The dual-targeting compound has broad activity against Gram-negative pathogens in vitro and in animal models of infection (Luther et al., 2019Luther A. Urfe