The CDC estimates that every year, at least2 million people are infected with drugresistant bacteria. Of these, 23,000 will

die. The financial cost to the US is expected torange from $20 billion to $35 billion. On a globalscale, the numbers are equally frightening. TheWorld Health Organization estimates 700,000people will die worldwide each year from multi-drug resistant micro-organisms. Premature deathscould reach 10 million annually by 2050, accord-ing to the United Kingdom’s Review onAntimicrobial Resistance. That could cost theglobal gross domestic product (GDP) $100 trillionin economic productivity.

Today, the alarm bell about antibiotic resistance issounding more loudly than ever, with several recentscares fresh in public officials’ minds (see Table 1).

Looking ahead, the problem of antibiotic resistantbacteria shows no sign of abating unless new strate-gies for developing novel antibiotics take hold.

New approaches to battling resistanceIn 2016, world leaders gathered at the UnitedNations, committed to act on antimicrobial resis-tance, promising that individual nations woulddevelop action plans to combat the problem.Earlier, the US Congress passed the GeneratingAntibiotic Incentives Now (GAIN) Act, creatingspecial designations to speed up new-drugapprovals and extending drug company patent

exclusivity by five years. The 21st Century CuresAct of 2016 further clears the pathway to patientswith new FDA approval schemes for serious or life-threatening infections. This provision is expectedto speed approval of new antibiotics, especially formulti-drug resistant micro-organisms.

Other government groups have acted moredirectly, investing in early development of newantibiotic molecules and strategies. The USNational Action Plan on Combatting Antibiotic-Resistant Bacteria (CARB) launched its accelerator,CARB-X, in 2016 with $500 million to jumpstartantibiotic development. CARB-X released its firsttranche of $48 million to 11 biopharmaceuticalcompanies in 2017. Similarly the BiomedicalAdvanced Research and Development Authority(BARDA), part of the US Department of Healthand Human Welfare (DHHS), has committed up to$250 million over five years to support new antibi-otic development.

Acknowledging that there have been no newantibiotics developed in the past 30 years, theUnited Kingdom established the AntimicrobialResistance Centre (AMR Centre) to support start-up work with $236 million with a goal of moving20 new medicines into pre-clinical development by2020 and advance 10 of those to clinical trials by2022. The AMR Centre recently issued a call toaction to the G20, an international forum for thegovernments and central bank governors from 20

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Tackling multi-drugresistant bacteriaAntibiotic resistance never should have become the public health crisis we areseeing today. As a society, we thought the problem of bacterial infections hadbeen solved decades ago when more than 200 powerful antibioticsrepresenting different classes such as �-lactam, tetracyclines and macrolideswere developed. Names such as amoxicillin, clindamycin and erythromycin havebecome well-known among consumers. Signs of resistance to antibioticsappeared almost immediately and continues to plague our healthcare systems.

major economies, to commit investment inresearch, innovation and development of innova-tive health technologies to counter threats fromantimicrobial resistance.

To better diagnose the problem, the USNational Institutes of Health (NIH) completed thefirst phase of its Antimicrobial ResistanceDiagnostic Challenge, a federal competition thatwill award up to $20 million in prizes for thedevelopment of rapid, point-of-need diagnostictests to combat drug-resistant bacteria. Ten semi-finalist were selected and each received $50,000 todevelop their concepts into prototypes.

While policy initiatives and government fundingare a major step forward, they will not be sufficientto solve this problem. In its Scientific Roadmap forAntibiotic Discovery, The Pew Charitable Trustsconcluded that the biggest barriers to finding newclasses of antibiotics are not regulatory or finan-cial: they are scientific.

New science needed There has not been a novel antibiotic classapproved for use against Gram-negative infectionsin the last 40 years. However, the lack of novelty isby no means an indication of lack of effort devotedto this cause.

Across the public, private and academic industries

many extensive drug discovery endeavours have beenundertaken, only to come up short of a novel thera-peutic. The reason for these numerous failures lies inthe inability to get good inhibitors to their targets inGram-negative bacteria. The structure of a bacte-ria’s cell wall determines whether it is Gram-positiveor Gram-negative (Figure 1). The Gram-positive cellwall consists of a thick peptidoglycan layer and aphospholipid membrane, while Gram-negative bac-teria possess an inner membrane consisting of phos-pholipid, a thin peptidoglycan layer, plus an outermembrane. The outer membrane of Gram-negativebacteria is an asymmetric bilayer composed of glyc-erol phospholipids and lipopolysaccharide (LPS).LPS acts as a barrier against toxic compounds,including antibiotics, whose targets typically residewithin the inner membrane. It is not surprising thatGram-negative bacteria cause a wide spectrum ofdiseases, including urinary tract, bloodstream, air-way, venereal and healthcare-associated infections.Both CDC and WHO have singled out Gram-nega-tive bacteria for special attention, as most antibioticswork well against Gram-positive bacteria, but notGram-negative.

In a 2016 study update, the Pew CharitableTrusts surveyed antibiotics currently in clinicaldevelopment and made a disheartening prediction:of the dozens of antibiotics in clinical development,only one in five are expected to be approved foruse. Despite the seeming doom and gloom, a num-ber of novel strategies have begun to emerge forcombating antibiotic resistant bacteria, especiallyfor those of the Gram-negative type. The approach-es can be grouped into a number of categoriesincluding activating existing antibiotics, indirecttherapeutics, biologics and small molecules.

Activating existing antibioticsGram-negative bacteria have a protective outerbarrier and fewer internal defences commonamong the Gram-positive bacteria, such asenzymes that degrade antibiotics or mechanismsthat pump antibiotics back out of the cell (efflux).Breeching the outer barrier makes Gram-negativesvulnerable to a wide range of existing antibiotics.

A company in the US, Spero Therapeutics, isdeveloping a series of novel chemical entities –Potentiators – that specifically interact with theouter membrane of Gram-negative bacteria to dis-rupt and increase the membrane’s permeability(Figure 2). This increase in permeability allowsboth novel and existing Gram-positive antibioticsto enter and kill the cell.

The first Potentiator candidate, SPR741, aderivative of polymyxin B currently being evaluated

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DATE EVENT

August 2016 A Nevada woman died from a carbapenem-resistantEnterobacteriaceae infection resistant to 26 differentantibiotics – essentially every antibiotic available tohealthcare providers.

December 2016 The University of California Irvine Medical Centeracknowledged that 10 infants contracted methicillin-resistant Staphylococcus aureus (MRSA) in its neonatalintensive care unit. Two months earlier, the World HealthOrganization had declared MRSA to be in one of a dozenfamilies of super bacteria that “pose the greatest threatto human health.”

April 2016 A Pennsylvania woman was found to be carrying a strainof E. coli resistant to colistin, an antibiotic of “last resort”,heralding the “emergence of a truly pan-drug resistantbacteria”, according to the doctors who discovered it.

2005-2016 Between 2005 and 2016, multidrug-resistant gramnegative bacteria (MDR-GNB), such as Escherichia coli,were found in more than a quarter of nursing homeresidents on average, according to a meta-analysispublished in the American Journal of Infection Control.

Table 1: Antibiotic-resistant bacteria in the news

in a Phase I clinical trial, sensitises Gram-negativebacteria to antibiotics normally excluded by theouter membrane.

Prior to initiating our clinical trial, we used bac-terial cytological profiling (BCP), a microscopy-based technique that provides insight into themechanism of action for antibacterial compounds,to define how SPR741 enhances sensitivity ofGram-negative bacteria to Gram-positive antibi-otics. In this study, we assessed SPR741 alone andin combination with fluorescently-labelledazithromycin (a macrolide) or Bocillin (a peni-

cillin), to observe uptake into Escherichia coli.Cytological profiling and cytometry results demon-strated that SPR741 permeabilises the outer mem-brane of Gram-negative bacteria, such as E.coli,allowing normally impenetrant antibacterials intothe periplasm and cytoplasm (Figure 3).

Preclinical studies show that by disrupting theouter membrane, SPR741 enables more than two-dozen classes of existing antibiotics normally onlyeffective against Gram-positive bacteria to enterand destroy the now-relatively defenceless Gram-negative bacteria.

Figure 1Comparison of the cell wallsof Gram-negative and Gram-positive bacteria. Therelatively-impermeable cellwall of Gram-negative bacteriarenders the organisms moreresistant to antibiotics

Figure 2Potentiator moleculesspecifically interact with theouter membrane of Gram-negative bacteria to increasethe membrane’s permeability,allowing Gram-positiveantibiotics to enter and kill the cell

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Figure 4 shows results from a study of the invitro efficacy of combinations of SPR741 with con-ventional antibiotics against Escherichia coli (Ec),Klebsiella pneumoniae (Kp), and Acinetobacterbaumannii (Ab). Of 35 antibiotics tested, the min-imum inhibitory concentration (MIC) of eight –azithromycin, clarithromycin (CLR), erythromycin(ERY), fusidic acid (FA), mupirocin, retapamulin(RET) rifampicin (RIF) and telithromycin – againstEc and Kp was reduced 32 – 8,000-fold in the pres-ence of 8-16µg/mL SPR741; against Ab, similarpotentiation was achieved with CLR, ERY, FA andRIF. SPR741 was able to potentiate antibiotics thatare substrates of the multi-drug efflux pumpAcrAB-TolC, effectively circumventing the pump’scontribution to intrinsic antibiotic resistance.Green arrows indicate that the MIC of the antibi-otic has been reduced to, or below, the Clinical andLaboratory Standards Institute (CLSI) or EuropeanCommittee on Antimicrobial Susceptibility Testing(EUCAST) breakpoint for susceptibility for thisorganism/antibiotic combination, or where thisinformation is not available, below the equivalentbreakpoint or epidemiological cut-off values(ECOFF) for Staphylococcus spp.

Indirect therapeuticsInstead of killing the bacteria directly, some strate-gies focus on blocking the bacteria’s virulence. Forexample, Microbiotix, Inc has developed a strategythat interferes with the Type III secretion system ofPseudomonas aeruginosa, a bacteria which pro-duces a toxin that plays a major role in ventilator-assisted pneumonia. Preventing release of the toxinshould block disease progression.

Biological strategiesThe war between the microbes has raged for giga-millennia. No battle is fiercer than the strugglebetween bacteria and specialised viruses that preyon them called bacteriophage. To release theirprogeny from inside an infected bacterial cell,phage encode a number of products called lysinsthat disrupt the bacterial cell wall, rupturing it andkilling the bacteria. The idea to use bacteriophagelysins as a medicine has been around for decades,but the advent and success of antibiotics shelvedmost projects.

With the rise of multi-drug resistance, lysins areexperiencing a renaissance. ContraFect Corpora-tion, for example, has identified 48 lysins that candirectly bind and disrupt the cell wall of Gram-neg-ative bacteria, killing them on contact. The compa-ny’s first bacteriophage-derived lysin has enteredclinical trials.

Another biological strategy relies on monoclonalantibodies (mAbs). Just as cancer researchers havesuccessfully engineered antibodies to seek out andkill cancer cells, scientists are now turning thosetools toward multi-drug resistant bacteria. Researchteams focused on developing novel mAbs againstmicro-organisms have begun to make progress. Inone interesting approach, Visterra Inc developed amonoclonal antibody that specifically targetsPseudomonas, and to which is chemically linked abactericidal peptide that directly kills the bacteria.

Small molecule discoveryQuite a number of biopharma companies pursuedifferent strategies to discover new antibiotics inthe traditional way. Tetraphase Pharmaceuticals

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Figure 3: Fluorescent microscopy show that E. coliATCC 25922 is largely impermeable to fluorescently-labelled azithromycin or Bocillin. Addition of 8μg/mLSPR741 increased the uptake of azithromycin and increased accumulation of fluorescent Bocillin. Pogliano J. ‘Bacterial Cytological Profiling of SPR741 Mechanismof Action is Consistent with Membrane Permeabilization that Allows Penetration of Antibiotics into Gram-negative (G-) Bacteria’. ASM Microbe 2016

Therapeutics

Inc uses a proprietary chemistry technology to cre-ate novel antibiotics. Oppliotech Ltd uses compu-tational modelling to develop its new agents.

Many of the drug development strategies fallalong similar lines and target specific parts of thebacteria. Forge Therapeutics, Inc is developingsmall molecules that target a metalloenzyme(LpxC) unique to bacteria. Redx Pharma Plc,MerLion Pharmaceuticals Pte Ltd and TaiGenBiotechnology Co Ltd all target novel bacterialtopoisomerases; enzymes critical to the replicationand transcription of bacterial DNA. Candidatemolecules from MicuRx Pharmaceuticals Inc,Actelion Pharmaceuticals Ltd and Wockhardt Ltddisrupt a specific bacterial ribosome; cellular struc-tures where protein synthesis occurs.

The way forward Bacteria have been evolving for billions of years andhave a head start on us. A study of ancient bacteriarecovered from Arctic permafrost found antibioticresistance genes existed tens of thousands of yearsago, having evolved to defend bacteria against theantibiotics made by other micro-organisms.

After decades of a relatively more relaxed viewin the fight against antibiotic-resistant bacteria,government and industry have a renewed, moreaggressive focus. Most of the antibacterialmolecules in use today came from microbes them-selves and may be considered the low hangingfruit. Scientists isolated antibiotics from nature andchemically tweaked them to produce related class-es of drugs. Fortunately, innovative strategies toattack bacteria are being pursued, giving us hopethat the tide may be ready to turn in the battleagainst antibiotic resistance. DDW

Dr Ankit Mahadevia is the CEO of SperoTherapeutics. He was formerly a Venture Partnerin the life sciences group at Atlas Venture. In thatcapacity he supported the formation of eight com-panies focused on novel drug discovery platformsand therapeutic products, three of which he led asActing CEO.

Figure 4SPR741 potentiates the activity of antibiotics against E.coli ATCC 25922 (A), K. pneumoniae ATCC 43816 (B)and A. baumannii NCTC 12156 (C). The fold reduction inthe MIC of each of 35 antibiotics is displayed at each ofthree concentrations of the Potentiator (2, 4, or 8µg/mL[A]; 4, 8, or 16µg/mL [B and C]). Corbett D.‘Potentiation of Antibiotic Activity by a Novel CationicPeptide, SPR741’. ASM Microbe 2016

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